Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment

BACKGROUNDTo solve the problem of liver transplantation donor insufficiency, an alternative cell transplantation therapy was investigated. We focused on amniotic epithelial cells (AECs) as a cell source because, unlike induced pluripotent stem cells, they are cost-effective and non-tumorigenic. The utilization of AECs in regenerative medicine, however, is in its infancy. A general profile for AECs has not been comprehensively analyzed. Moreover, no hepatic differentiation protocol for AECs has yet been established. To this end, we independently compiled human AEC libraries, purified amniotic stem cells (ASCs), and co-cultured them with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cell (HUVECs) in a 3D system which induces functional hepatic organoids.AIMTo characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cellsMETHODSAECs, MSCs, and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients. Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry, Alkaline phosphatase (AP) staining, and flow cytometry. An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay. AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics. The 2D and 3D culture were compared by relative gene expression using several differentiation protocols. ASCs, MSCs, and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging, periodic acid Schiff, and an indocyanine green (ICG) test.RESULTSAECs have certain stemness markers such as EPCAM, SSEA4, and E-cadherin. One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers. Moreover, it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage. Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells. TJP1, associated with epidermal growth factor receptor, and MET, associated with hepatocyte growth factor receptor, were upregulated and may be important for hepatic differentiation. In conventional flat culture, the cells turned unviable and did not readily differentiate into hepatocytes. In 3D culture, however, hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol. Furthermore, the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity, hepatic-like glycogen storage, and ICG absorption/elimination.CONCLUSIONHuman amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness. Under a 3D co-culture system, functional hepatic organoids were generated in a multicellular microenvironment

The Blimp1–Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis

Controlling osteoclastogenesis is critical to maintain physiological bone homeostasis and prevent skeletal disorders. Although signaling activating nuclear factor of activated T cells 1 (NFATc1), a transcription factor essential for osteoclastogenesis, has been intensively investigated, factors antagonistic to NFATc1 in osteoclasts have not been characterized. Here, we describe a novel pathway that maintains bone homeostasis via two transcriptional repressors, B cell lymphoma 6 (Bcl6) and B lymphocyte–induced maturation protein-1 (Blimp1). We show that Bcl6 directly targets ‘osteoclastic’ molecules such as NFATc1, cathepsin K, and dendritic cell-specific transmembrane protein (DC-STAMP), all of which are targets of NFATc1. Bcl6-overexpression inhibited osteoclastogenesis in vitro, whereas Bcl6-deficient mice showed accelerated osteoclast differentiation and severe osteoporosis. We report that Bcl6 is a direct target of Blimp1 and that mice lacking Blimp1 in osteoclasts exhibit osteopetrosis caused by impaired osteoclastogenesis resulting from Bcl6 up-regulation. Indeed, mice doubly mutant in Blimp1 and Bcl6 in osteoclasts exhibited decreased bone mass with increased osteoclastogenesis relative to osteoclast-specific Blimp1-deficient mice. These results reveal a Blimp1–Bcl6–osteoclastic molecule axis, which critically regulates bone homeostasis by controlling osteoclastogenesis and may provide a molecular basis for novel therapeutic strategies

Higher D-dimer level in the early third trimester predicts the occurrence of postpartum hemorrhage

Aims: This study aimed to determine effective predictive factors for primary postpartum hemorrhage (PPH) among clinical blood parameters associated with coagulation and fibrinolysis and demographic characteristics.Methods: We retrospectively studied 1032 women who underwent determinations of clinical blood parameters at gestational week (GW) 29–32 and GW 35–37 and gave birth to singleton infants at our hospital between January 2011 and December 2013. PPH was defined as estimated blood loss ≥700 mL. Multivariate logistic regression analyses were used to determine independent risk factors and odds ratios (OR) for PPH.Results: PPH occurred in 104 of 1032 women (10%). Three blood variables, fibrinogen level 2.7 μg/mL (2.03 [1.29–3.19]) at GW 35–37, and three demographic characteristics, maternal age ≥35 years (1.75 [1.15–2.68]), BMI >28.2 kg/m2 on admission for childbirth (1.95 [1.20–3.16]), and previous cesarean delivery (2.77 [1.31–5.83]), were identified as independent risk factors for PPH.Conclusion: Among blood parameters, higher D-dimer levels and lower levels of antithrombin activity and fibrinogen in late gestation were independent risk factors for PPH

Supersaturation-limited amyloid fibrillation of insulin revealed by ultrasonication

Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. We proposed that ultrasonication may be an effective agitation to trigger nucleation that would otherwise not occur under the persistent metastability of supersaturation. However, the roles of supersaturation and effects of ultrasonication have not been elucidated in detail except for limited cases. Insulin is an amyloidogenic protein that is useful for investigating the mechanisms underlying amyloid fibrillation with biological relevance. We studied the alcohol-induced amyloid fibrillation of insulin using various concentrations of 2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoro-2-propanol at pH 2.0 and 4.8. Ultrasonic irradiation effectively triggered fibrillation under conditions in which insulin retained persistent supersaturation. Structural analyses by circular dichroism, Fourier transform infrared spectroscopy, transmission electron microscopy, and atomic force microscopy revealed that the dominant structures of fibrils varied between parallel and antiparallel β-sheets depending on the solvent conditions. pH and alcohol concentration-dependent phase diagrams showed a marked difference before and after the ultrasonic treatment, which indicated that the persistent metastability of supersaturation determined the conformations of insulin. These results indicate the importance of an alternative view of amyloid fibrils as supersaturation-limited crystal-like aggregates formed above the solubility limit.This research was originally published in the Journal of Biological Chemistry. Hiroya Muta, Young-Ho Lee, József Kardos, Yuxi Lin, Hisashi Yagi and Yuji Goto. Supersaturation-limited Amyloid Fibrillation of Insulin Revealed by Ultrasonication. J. Biol. Chem. 2014; 289, 18228–18238. © the American Society for Biochemistry and Molecular Biolog