Human archetypal pluripotent stem cells differentiate into trophoblast stem cells via endogenous BMP5/7 induction without transitioning through naive state.

Primary human trophoblast stem cells (TSCs) and TSCs derived from human pluripotent stem cells (hPSCs) can potentially model placental processes in vitro. Yet, the pluripotent states and factors involved in the differentiation of hPSCs to TSCs remain poorly understood. In this study, we demonstrate that the primed pluripotent state can generate TSCs by activating pathways such as Epidermal Growth Factor (EGF) and Wingless-related integration site (WNT), and by suppressing tumor growth factor beta (TGFß), histone deacetylases (HDAC), and Rho-associated protein kinase (ROCK) signaling pathways, all without the addition of exogenous Bone morphogenetic protein 4 (BMP4)-a condition we refer to as the TS condition. We characterized this process using temporal single-cell RNA sequencing to compare TS conditions with differentiation protocols involving BMP4 activation alone or BMP4 activation in conjunction with WNT inhibition. The TS condition consistently produced a stable, proliferative cell type that closely mimics first-trimester placental cytotrophoblasts, marked by the activation of endogenous retroviral genes and the absence of amnion expression. This was observed across multiple cell lines, including various primed induced pluripotent stem cell (iPSC) and embryonic stem cell (ESC) lines. Primed-derived TSCs can proliferate for over 30 passages and further specify into multinucleated syncytiotrophoblasts and extravillous trophoblast cells. Our research establishes that the differentiation of primed hPSCs to TSC under TS conditions triggers the induction of TMSB4X, BMP5/7, GATA3, and TFAP2A without progressing through a naive state. These findings propose that the primed hPSC state is part of a continuum of potency with the capacity to differentiate into TSCs through multiple routes.

[Family's Perception of Proxy Decision Making to Authorize Do Not Resuscitate Order of Elderly Patients in Long Term Care Facility: A Q-Methodological Study].

PURPOSE: This study aimed to distinguish and describe the types of perceptions of do not resuscitate (DNR) proxy decisions among families of elderly patients in a long-term care facility. METHODS: This exploratory study applied Q-methodology, which focuses on individual subjectivity. Thirty-four Q-statements were selected from 130 Q-populations formed based on the results of in-depth interviews and literature reviews. The P-samples were 34 families of elderly patients in a long-term care hospital in Busan, Korea. They categorized the Q-statements using a 9-point scale. Using the PC-QUANL program, factor analysis was performed with the P-samples along an axis. RESULTS: The families' perceptions of the DNR proxy decision were categorized into three types. Type I, rational acceptance, valued consensus among family members based on comprehensive support from medical staff. Type II, psychological burden, involved hesitance in making a DNR proxy decision because of negative emotions and psychological conflict. Type III, discreet decisions, valued the patients' right to self-determination and desire for a legitimate proxy decision. Type I included 18 participants, which was the most common type, and types II and III each included eight participants. CONCLUSION: Families' perceptions of DNR proxy decisions vary, requiring tailored care and intervention. We suggest developing and providing interventions that may psychologically support families.

Novel culture system via wirelessly controllable optical stimulation of the FGF signaling pathway for human and pig pluripotency.

Stem cell fate is largely determined by cellular signaling networks and is heavily dependent on the supplementation of exogenous recombinant proteins into culture media; however, uneven distribution and inconsistent stability of recombinant proteins are closely associated with the spontaneous differentiation of pluripotent stem cells (PSCs) and result in significant costs in large-scale manufacturing. Here, we report a novel PSC culture system via wirelessly controllable optical activation of the fibroblast growth factor (FGF) signaling pathway without the need for supplementation of recombinant FGF2 protein, a key molecule for maintaining pluripotency of PSCs. Using a fusion protein between the cytoplasmic region of the FGF receptor-1 and a light-oxygen-voltage domain, we achieved tunable, blue light-dependent activation of FGF signaling in human and porcine PSCs. Our data demonstrate that a highly controllable optical stimulation of the FGF signaling pathway is sufficient for long-term maintenance of PSCs, without the loss of differentiation potential into three germ layers. This culture system will be a cost-effective platform for a large-scale stem cell culture.

Transcriptional landscape of myogenesis from human pluripotent stem cells reveals a key role of TWIST1 in maintenance of skeletal muscle progenitors.

Generation of skeletal muscle cells with human pluripotent stem cells (hPSCs) opens new avenues for deciphering essential, but poorly understood aspects of transcriptional regulation in human myogenic specification. In this study, we characterized the transcriptional landscape of distinct human myogenic stages, including OCT4::EGFP+ pluripotent stem cells, MSGN1::EGFP+ presomite cells, PAX7::EGFP+ skeletal muscle progenitor cells, MYOG::EGFP+ myoblasts, and multinucleated myotubes. We defined signature gene expression profiles from each isolated cell population with unbiased clustering analysis, which provided unique insights into the transcriptional dynamics of human myogenesis from undifferentiated hPSCs to fully differentiated myotubes. Using a knock-out strategy, we identified TWIST1 as a critical factor in maintenance of human PAX7::EGFP+ putative skeletal muscle progenitor cells. Our data revealed a new role of TWIST1 in human skeletal muscle progenitors, and we have established a foundation to identify transcriptional regulations of human myogenic ontogeny (online database can be accessed in http://www.myogenesis.net/).

Noninvasive Assessment of Exosome Pharmacokinetics In Vivo: A Review.

Exosomes, intraluminal vesicles that contain informative DNA, RNA, proteins, and lipid membranes derived from the original donor cells, have recently been introduced to therapy and diagnosis. With their emergence as an alternative to cell therapy and having undergone clinical trials, proper analytical standards for evaluating their pharmacokinetics must now be established. Molecular imaging techniques such as fluorescence imaging, magnetic resonance imaging, and positron emission tomography (PET) are helpful to visualizing the absorption, distribution, metabolism, and excretion of exosomes. After exosomes labelled with a fluorescer or radioisotope are administered in vivo, they are differentially distributed according to the characteristics of each tissue or lesion, and real-time biodistribution of exosomes can be noninvasively monitored. Quantitative analysis of exosome concentration in biological fluid or tissue samples is also needed for the clinical application and industrialization of exosomes. In this review, we will discuss recent pharmacokinetic applications to exosomes, including labelling methods for in vivo imaging and analytical methods for quantifying exosomes, which will be helpful for evaluating pharmacokinetics of exosomes and improving exosome development and therapy.

TRRUST: a reference database of human transcriptional regulatory interactions.

The reconstruction of transcriptional regulatory networks (TRNs) is a long-standing challenge in human genetics. Numerous computational methods have been developed to infer regulatory interactions between human transcriptional factors (TFs) and target genes from high-throughput data, and their performance evaluation requires gold-standard interactions. Here we present a database of literature-curated human TF-target interactions, TRRUST (transcriptional regulatory relationships unravelled by sentence-based text-mining, http://www.grnpedia.org/trrust), which currently contains 8,015 interactions between 748 TF genes and 1,975 non-TF genes. A sentence-based text-mining approach was employed for efficient manual curation of regulatory interactions from approximately 20 million Medline abstracts. To the best of our knowledge, TRRUST is the largest publicly available database of literature-curated human TF-target interactions to date. TRRUST also has several useful features: i) information about the mode-of-regulation; ii) tests for target modularity of a query TF; iii) tests for TF cooperativity of a query target; iv) inferences about cooperating TFs of a query TF; and v) prioritizing associated pathways and diseases with a query TF. We observed high enrichment of TF-target pairs in TRRUST for top-scored interactions inferred from high-throughput data, which suggests that TRRUST provides a reliable benchmark for the computational reconstruction of human TRNs.

RANTES polymorphisms and the risk of graft-versus-host disease in human leukocyte antigen-matched sibling allogeneic hematopoietic stem cell transplantation.

We investigated the association between RANTES (regulated upon activation, normal T cell expressed and secreted) polymorphisms and clinical outcomes in patients treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Three RANTES gene polymorphisms, i.e., -403G/A (rs2107538), -28C/G (rs2280788) and In1.1T/C (rs2280789), were genotyped, and the effects of the genotypes and haplotypes of RANTES on clinical outcomes were analyzed. The competing risk regression analysis was used to investigate the relationship between the polymorphisms and the cumulative risk of graft-versus-host disease (GVHD). An AGC haplotype in a recessive model showed significant harmful effects on the cumulative risk of acute GVHD and relapse-free survival (adjusted hazard ratios 2.42 and 2.71, 95% confidence intervals 1.29-4.55 and 1.30-5.64; p = 0.018 and 0.024, respectively), whereas a GCT haplotype did not. RANTES polymorphisms were not significantly associated with overall survival and the risk of chronic GVHD. This study suggests that RANTES polymorphisms might be associated with the occurrence of acute GVHD rather than of chronic GVHD and also of relapse-free survival in the patients treated with allo-HSCT. Further larger prospective investigations are needed to establish the role of RANTES polymorphisms in patients treated with allo-HSCT.

Impact of vitamin D receptor gene polymorphisms on clinical outcomes of HLA-matched sibling hematopoietic stem cell transplantation.

We hypothesized that polymorphisms of the vitamin D receptor (VDR) gene might affect clinical outcomes of allogeneic hematopoietic stem cell transplantation (HSCT). Three VDR gene polymorphisms (BsmI G>A, ApaI G>T, and TaqI T>C) were genotyped in 147 patients who underwent HLA-matched sibling allogeneic HSCT. Frequencies of infection, graft-vs.-host disease (GVHD), overall survival (OS), and disease-free survival (DFS) were compared according to genotypes and haplotypes. Infection and acute GVHD had trends to be less frequent in patients with ApaI TT genotype than non-TT genotypes (p = 0.061 and p = 0.059, respectively). For TaqI genotypes, there were no statistical differences in frequency of infection and acute GVHD (p = 0.84 and p = 0.30, respectively), but TC genotype was associated with longer OS and DFS than TT genotype (p = 0.022 and p = 0.038, respectively). In the ApaI-TaqI haplotype analysis, patients with TC haplotype had significantly longer OS and DFS than those without TC haplotype (p = 0.022 and p = 0.038, respectively). In multivariable analysis, TaqI genotype and ApaI-TaqI haplotype of recipients were independent prognostic factors for both OS and DFS. This study suggests that the genotype and haplotype of VDR in recipient might be associated with clinical outcome of sibling HLA-matched HSCT.

Patient HSP70-hom TG haplotype is associated with decreased transplant-related mortality and improved survival after sibling HLA-matched hematopoietic stem cell transplantation.

Heat shock protein 70-hom (HSP70-hom) plays an important role in protein folding and immune responses. Therefore, HSP70-hom gene polymorphisms may act as important factors in predicting the prognosis of patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). To evaluate the role of HSP70-hom gene polymorphisms in the prognosis of patients receiving sibling human leukocyte antigen (HLA)-matched allogeneic HSCT, the HSP70-hom polymorphisms, T2437C and G2763A, were genotyped in 147 patients receiving sibling HLA-matched allogeneic HSCT. Individual diplotypes were estimated from genotype data of the two HSP70-hom polymorphisms using the expectation maximization algorithm. Patients with the 2763GG or GA genotype showed longer overall survival compared with those with the 2763AA genotype, and patients with a TG haplotype (TG/TA, TG/TG or TG/CG) also showed longer overall survival compared with those with a non-TG haplotype (TA/TA or TA/CG) (both G2763A genotype and diplotype, p<0.01). Moreover, the 2437TT genotype was found to be protective for treatment-related death compared with the 2437TC genotype, and a TG haplotype was found to be very protective for treatment-related death compared with a non-TG haplotype (T2437C genotype, p=0.04; and diplotype, p=0.02). Therefore, our results suggest that HSP70-hom polymorphisms play an important role in the prognosis of patients receiving sibling HLA-matched allogeneic HSCT.

Carbonic anhydrase 9 is a predictive marker of survival benefit from lower dose of bevacizumab in patients with previously treated metastatic colorectal cancer.

BACKGROUND: Carbonic anhydrase 9 (CA9) is a marker for hypoxia and acidosis, which is linked to a poor prognosis in human tumors. The purpose of this comparative analysis was to evaluate whether CA9 and VEGF expression are associated with survival outcomes in patients with metastatic colorectal cancer (mCRC) after treatment with bevacizumab as second or later line treatment. METHODS: Thirty-one mCRC patients who were treated with bevacizumab-containing chemotherapy as second or later line treatment and who had analyzable tumor paraffin blocks were selected for this study. The planned dose of bevacizumab was 5 mg/kg/2-week. Immunohistochemical (IHC) staining of CA9 and VEGF was performed and their expression was scored by the intensity multiplied by percentage of stained area. RESULTS: The overall response rate was 19.4% and the disease control rate (DCR) was 61.3% with 6 partial responses and 13 cases of stable disease. The DCR was significantly higher in patients with a lower CA9 expression score compared to those with a higher score (80.0% vs. 27.3%, respectively, P = 0.004). The patients with a low CA9 expression score also showed better outcomes with regard to the median progression-free survival (P = 0.028) and overall survival (P = 0.026). However, VEGF expression was not associated with the DCR and survival. CONCLUSION: Lower degree of CA9 expression was associated with better clinical outcomes in patients with mCRC treated with lower dose bevacizumab-based chemotherapy. Prospective studies are now needed to determine the correlation between CA9 expression and clinical outcomes after bevacizumab treatment, at different doses and in varied settings.

A case of desmoplastic small round cell tumor diagnosed in a young female patient.

Desmoplastic small round cell tumor is a very rare malignancy. We report the case of a 26-year-old woman who suffered from dyspepsia and abdominal pain for 2 months. We performed an endoscopic biopsy of the duodenal mass and diagnosed her disease as desmoplastic small round cell tumor using immunohistochemical staining, fluorescence in situ hybridization, and reverse transcriptase polymerase chain reaction. Because the mass invaded the pancreas and superior mesenteric vein as well as duodenum and the disease was disseminated to liver and peritoneum, she received palliative chemotherapy using vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide. The maximal response to chemotherapy was stable disease. The patient expired 9 months after diagnosis.

Pathologic complete response after palliative 3rd line chemotherapy with capecitabine alone in metastatic colorectal cancer.

We report the case of a 46-year-old female who showed excellent clinical outcomes after palliative 3rd line capecitabine monotherapy followed by 2nd liver metastasectomy. She had been diagnosed with colon cancer with liver metastasis and initially treated with synchronous colectomy and liver metastasectomy. But she experienced immediate relapse in liver and received palliative 1st line FOLFIRI and 2nd line FOLFOX, both of which failed to show responses. Capecitabine alone as palliative 3rd line treatment did show clinical response in this patient, indeed, and she received 2nd liver metastasectomy which revealed pathologic complete response. In this report, we intended to emphasize that response to chemotherapy could be an important factor in patients who are potential candidates for metastasectomy and that catching the very moment of repeated metastasectomy is in the state of the art.