Profiling Insulin Like Factor 3 (INSL3) Signaling in Human Osteoblasts

Abstract BACKGROUND: Young men with mutations in the gene for the INSL3 receptor (Relaxin family peptide 2, RXFP2) are at risk of reduced bone mass and osteoporosis. Consistent with the human phenotype, bone analyses of Rxfp2(-/-) mice showed decreased bone volume, alterations of the trabecular bone, reduced mineralizing surface, bone formation, and osteoclast surface. The aim of this study was to elucidate the INSL3/RXFP2 signaling pathways and targets in human osteoblasts. METHODOLOGY/PRINCIPAL FINDINGS: Alkaline phosphatase (ALP) production, protein phosphorylation, intracellular calcium, gene expression, and mineralization studies have been performed. INSL3 induced a significant increase in ALP production, and Western blot and ELISA analyses of multiple intracellular signaling pathway molecules and their phosphorylation status revealed that the MAPK was the major pathway influenced by INSL3, whereas it does not modify intracellular calcium concentration. Quantitative Real Time PCR and Western blotting showed that INSL3 regulates the expression of different osteoblast markers. Alizarin red-S staining confirmed that INSL3-stimulated osteoblasts are fully differentiated and able to mineralize the extracellular matrix. CONCLUSIONS/SIGNIFICANCE: Together with previous findings, this study demonstrates that the INSL3/RXFP2 system is involved in bone metabolism by acting on the MAPK cascade and stimulating transcription of important genes of osteoblast maturation/differentiation and osteoclastogenesis

Long-term in vitro expansion of a human fetal pancreas stem cell that generates all three pancreatic cell lineages

The mammalian pancreas consists of three epithelial compartments: the acini and ducts of the exocrine pancreas and the endocrine islets of Langerhans. Murine studies indicate that these three compartments derive from a transient, common pancreatic progenitor. Here, we report derivation of 18 human fetal pancreas organoid (hfPO) lines from gestational weeks 8–17 (8–17 GWs) fetal pancreas samples. Four of these lines, derived from 15 to 16 GWs samples, generate acinar-, ductal-, and endocrine-lineage cells while expanding exponentially for >2 years under optimized culture conditions. Single-cell RNA sequencing identifies rare LGR5+ cells in fetal pancreas and in hfPOs as the root of the developmental hierarchy. These LGR5+ cells share multiple markers with adult gastrointestinal tract stem cells. Organoids derived from single LGR5+ organoid-derived cells recapitulate this tripotency in vitro. We describe a human fetal tripotent stem/progenitor cell capable of long-term expansion in vitro and of generating all three pancreatic cell lineages. Nephrolog

Human extrahepatic and intrahepatic cholangiocyte organoids show region-specific differentiation potential and model cystic fibrosis-related bile duct disease

The development, homeostasis, and repair of intrahepatic and extrahepatic bile ducts are thought to involve distinct mechanisms including proliferation and maturation of cholangiocyte and progenitor cells. This study aimed to characterize human extrahepatic cholangiocyte organoids (ECO) using canonical Wnt-stimulated culture medium previously developed for intrahepatic cholangiocyte organoids (ICO). Paired ECO and ICO were derived from common bile duct and liver tissue, respectively. Characterization showed both organoid types were highly similar, though some differences in size and gene expression were observed. Both ECO and ICO have cholangiocyte fate differentiation capacity. However, unlike ICO, ECO lack the potential for differentiation towards a hepatocyte-like fate. Importantly, ECO derived from a cystic fibrosis patient showed no CFTR channel activity but normal chloride channel and MDR1 transporter activity. In conclusion, this study shows that ECO and ICO have distinct lineage fate and that ECO provide a competent model to study extrahepatic bile duct diseases like cystic fibrosis

Long-term in vitro expansion of a human fetal pancreas stem cell that generates all three pancreatic cell lineages

The mammalian pancreas consists of three epithelial compartments: the acini and ducts of the exocrine pancreas and the endocrine islets of Langerhans. Murine studies indicate that these three compartments derive from a transient, common pancreatic progenitor. Here, we report derivation of 18 human fetal pancreas organoid (hfPO) lines from gestational weeks 8–17 (8–17 GWs) fetal pancreas samples. Four of these lines, derived from 15 to 16 GWs samples, generate acinar-, ductal-, and endocrine-lineage cells while expanding exponentially for >2 years under optimized culture conditions. Single-cell RNA sequencing identifies rare LGR5+ cells in fetal pancreas and in hfPOs as the root of the developmental hierarchy. These LGR5+ cells share multiple markers with adult gastrointestinal tract stem cells. Organoids derived from single LGR5+ organoid-derived cells recapitulate this tripotency in vitro. We describe a human fetal tripotent stem/progenitor cell capable of long-term expansion in vitro and of generating all three pancreatic cell lineages

Long-term culture of genome-stable bipotent stem cells from adult human liver

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy

Human extrahepatic and intrahepatic cholangiocyte organoids show region-specific differentiation potential and model cystic fibrosis-related bile duct disease

The development, homeostasis, and repair of intrahepatic and extrahepatic bile ducts are thought to involve distinct mechanisms including proliferation and maturation of cholangiocyte and progenitor cells. This study aimed to characterize human extrahepatic cholangiocyte organoids (ECO) using canonical Wnt-stimulated culture medium previously developed for intrahepatic cholangiocyte organoids (ICO). Paired ECO and ICO were derived from common bile duct and liver tissue, respectively. Characterization showed both organoid types were highly similar, though some differences in size and gene expression were observed. Both ECO and ICO have cholangiocyte fate differentiation capacity. However, unlike ICO, ECO lack the potential for differentiation towards a hepatocyte-like fate. Importantly, ECO derived from a cystic fibrosis patient showed n

Healing through culturally embedded practice: an investigation of counsellors’ and clients’ experiences of Buddhist Counselling in Thailand

This thesis is concerned with an exploration of counsellors’ and clients’ lived experiences of Buddhist Counselling, an indigenous Buddhist-based counselling approach in Thailand. Over the past decade, Buddhist Counselling has received a growing interest from Thai counselling trainees and practitioners, and it has also expanded to serve Thai people in various settings. Research on Buddhist Counselling is very limited and most of the existing studies in the field have focused on measuring the effectiveness of the approach. While these studies have consistently indicated the positive effects of Buddhist Counselling on psychological improvement across several population groups, the significant questions of how Buddhist Counselling brings about such outcome and how it is experienced are still largely unanswered. Moreover, existing research is concentrated much more on clients’ views than counsellors’ views, although counsellors’ views of their counselling practice can also serve as a knowledge base of the field. This thesis thus sets out to contribute to rectifying this omission by exploring Buddhist Counselling from the perspectives of both counsellors and clients. The thesis is based on two qualitative studies. The first study addressed Buddhist Counselling from the perspective of five counsellors through a focus group and semi-structured interviews. The second study explored Buddhist Counselling from the perspective of three clients, using two semi-structured interviews with each of them. All data received were analysed using interpretative phenomenological analysis (IPA). The study reveals counsellors’ and clients’ overall positive experience of engaging in Buddhist Counselling. Central to the accounts of the counsellors are the following perceptions: that their practice of Buddhist Counselling is culturally congruent with the existing values and beliefs of both themselves and their clients; that their personal and professional congruence is key to their therapeutic efficacy; and that they enhance such congruence through their application of Buddhist ideas and practices in their daily lives. Key to the clients’ accounts is their emphasis on the significant roles of the counsellors’ Buddhist ideas and personal qualities, and of their religious practices in facilitating healing and change. Key shared findings from both studies reveal that the participants’ accounts of their cultural background and their experiences of Buddhist Counselling are intertwined. Adopting hermeneutics to address this intertwinement, I reveal the cultural and moral dimensions underlying the practice of Buddhist Counselling. Based on such revelation, I suggest that Buddhist Counselling in particular, as well as psychotherapy in general, should be better understood as a historically situated, culturally bound, and morally constituted activity of people who are concerned with improving the quality of their lives and their community, rather than the transcultural and merely relational work of morally-neutral practitioners