19 research outputs found
Expression profiling in the characterization of differentiating human embryonic stem cells
No full textDuring Embryogenesis, the harbinger of germ layer formation is the primitive streak (PS), a transient structure oriented along the embryonic anteroposterior axis, through which epiblast cells egress to form mesoderm and endoderm. Hematopoietic and vascular lineages arise in multiple sites and at different times during embryogenesis from extra embryonic and lateral plate mesoderm, derivatives of the posterior and mid regions of the PS. In vitro differentiation of human embryonic stem cells (hESCs) has been demonstrated to recapitulate aspects of vertebrate development, and we have established an in vitro hESC differentiation system to study the initiation of gastrulation as well as the development of early hematopoietic mesoderm in a defined, serum-free medium, showing that exogenously added TGFb family molecules induce primitive streak-like cells that subsequently differentiate into mesodermal and endodermal lineages. In order to ascertain the precise isolation of mesodermal precursors, a targeted hESC line in which GFP was inserted by homologous recombination in the locus of the MIXL1 homeobox gene (MIXL1GFP/W) was generated and used as guidance for the identification of viable, PS-like, cells. In this study, microarray-based transcriptional profiling was used to examine gene expression during mesoderm induction from MIXL1GFP/W hESCs in isolated mesodermal populations. The aim of this study was to identify novel cell surface markers and growth factors that would facilitate the identification and generation of hematopoietic lineages from hESCs. During this study, transcriptional profiling method was also used to assist the characterization of hESCs derived cell phenotypes, such as neuronal, endothelial, cardiac and endodermal pancreatic progenitor cells
Expression profiling in the characterization of differentiating human embryonic stem cells
No full textDuring Embryogenesis, the harbinger of germ layer formation is the primitive streak (PS), a transient structure oriented along the embryonic anteroposterior axis, through which epiblast cells egress to form mesoderm and endoderm. Hematopoietic and vascular lineages arise in multiple sites and at different times during embryogenesis from extra embryonic and lateral plate mesoderm, derivatives of the posterior and mid regions of the PS.
In vitro differentiation of human embryonic stem cells (hESCs) has been demonstrated to recapitulate aspects of vertebrate development, and we have established an in vitro hESC differentiation system to study the initiation of gastrulation as well as the development of early hematopoietic mesoderm in a defined, serum-free medium, showing that exogenously added TGFb family molecules induce primitive streak-like cells that subsequently differentiate into mesodermal and endodermal lineages. In order to ascertain the precise isolation of mesodermal precursors, a targeted hESC line in which GFP was inserted by homologous recombination in the locus of the MIXL1 homeobox gene (MIXL1GFP/W) was generated and used as guidance for the identification of viable, PS-like, cells.
In this study, microarray-based transcriptional profiling was used to examine gene expression during mesoderm induction from MIXL1GFP/W hESCs in isolated mesodermal populations. The aim of this study was to identify novel cell surface markers and growth factors that would facilitate the identification and generation of hematopoietic lineages from hESCs. During this study, transcriptional profiling method was also used to assist the characterization of hESCs derived cell phenotypes, such as neuronal, endothelial, cardiac and endodermal pancreatic progenitor cells
Author Correction: Hormones induce the formation of luminal-derived basal cells in the mammary gland
No full textIn the initial published version of this article, there was a mistake in one author name (Jingsong Li). The correct name should be “Jinsong Li”. This correction does not affect the description of the results or the conclusions of this work.</p
A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells
No full textEndothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.</p
Identification of blood vascular endothelial stem cells by the expression of protein C receptor
No full textVascular growth and remodeling are dependent on the generation of new endothelial cells from stem cells and the involvement of perivascular cells to maintain vessel integrity and function. The existence and cellular identity of vascular endothelial stem cells (VESCs) remain unclear. The perivascular pericytes in adult tissues are thought to arise from the recruitment and differentiation of mesenchymal progenitors during early development. In this study, we identified Protein C receptor-expressing (Procr +) endothelial cells as VESCs in multiple tissues. Procr + VESCs exhibit robust clonogenicity in culture, high vessel reconstitution efficiency in transplantation, long-term clonal expansion in lineage tracing, and EndMT characteristics. Moreover, Procr + VESCs are bipotent, giving rise to de novo formation of endothelial cells and pericytes. This represents a novel origin of pericytes in adult angiogenesis, reshaping our understanding of blood vessel development and homeostatic process. Our study may also provide a more precise therapeutic target to inhibit pathological angiogenesis and tumor growth.</p
The role of endothelial cells in pancreatic islet development, transplantation and culture
No full textEndothelial cells (ECs) play pivotal roles in the development and maintenance of tissue homeostasis. During development, vasculature actively involves in organ morphogenesis and functional maturation, through the secretion of angiocrine factors and extracellular matrix components. Islets of Langerhans, essential functional units of glucose homeostasis, are embedded in a dense endothelial capillary network. Islet vasculature not only supplies nutrients and oxygen to endocrine cells but also facilitate the rapid delivery of pancreatic hormones to target tissues, thereby ensuring precise glucose regulation. Diabetes mellitus is a major disease burden and is caused by islet dysfunction or depletion, often accompanied by vessel loss and dysregulation. Therefore, elucidating the regulatory mechanisms of ECs within islets hold profound implications for diabetes therapy. This review provides an overview of recent research advancements on the functional roles of ECs in islet biology, transplantation, and in vitro islet organoid culture
Mammary Development and Breast Cancer: A Wnt Perspective
No full textThe Wnt pathway has emerged as a key signaling cascade participating in mammary organogenesis and breast oncogenesis. In this review, we will summarize the current knowledge of how the pathway regulates stem cells and normal development of the mammary gland, and discuss how its various components contribute to breast carcinoma pathology
Angiotensin II induces soluble fms-Like tyrosine kinase-1 release via calcineurin signaling pathway in pregnancy
No full textMaternal endothelial dysfunction in preeclampsia is associated with increased soluble fms-like tyrosine kinase-1 (sFlt-1), a circulating antagonist of vascular endothelial growth factor and placental growth factor. Angiotensin II (Ang II) is a potent vasoconstrictor that increases concomitant with sFlt-1 during pregnancy. Therefore, we speculated that Ang II may promote the expression of sFlt-1 in pregnancy. Here we report that infusion of Ang II significantly increases circulating levels of sFlt-1 in pregnant mice, thereby demonstrating that Ang II is a regulator of sFlt-1 secretion in vivo. Furthermore, Ang II stimulated sFlt-1 production in a dose- and time-dependent manner from human villous explants and cultured trophoblasts but not from endothelial cells, suggesting that trophoblasts are the primary source of sFlt-1 during pregnancy. As expected, Ang II-induced sFlt-1 secretion resulted in the inhibition of endothelial cell migration and in vitro tube formation. In vitro and in vivo studies with losartan, small interfering RNA specific for calcineurin and FK506 demonstrated that Ang II-mediated sFlt-1 release was via Ang II type 1 receptor activation and calcineurin signaling, respectively. These findings reveal a previously unrecognized regulatory role for Ang II on sFlt-1 expression in murine and human pregnancy and suggest that elevated sFlt-1 levels in preeclampsia may be caused by a dysregulation of the local renin/angiotensin system
Amphiregulin mediates the hormonal regulation on Rspondin-1 expression in the mammary gland
No full textThe steroid hormones are instrumental for the growth of mammary epithelial cells. Our previous study indicates that hormones regulate the expression of Rspondin-1 (Rspo1). Yet, the regulatory mechanism remains unknown. In the current study, we identify Amphiregulin (Areg) as a novel upstream regulator of Rspo1 expression mediating the hormonal influence. In response to hormonal signaling, Areg emanating from estrogen receptor (ER)-positive luminal cells, induce the expression of Rspo1 in ER-negative luminal cells. The paracrine action of Areg on Rspo1 expression is dependent on Egfr. Our data reveal a novel Estrogen-Areg-Rspo1 regulatory axis in the mammary gland, providing new evidence for the orchestrated action of systemic hormones and local growth factors.</p
Lentiviral CRISPR-guided RNA library screening identified Adam17 as an upstream negative regulator of Procr in mammary epithelium
No full textBackground: Protein C receptor (Procr) has recently been shown to mark resident adult stem cells in the mammary gland, vascular system, and pancreatic islets. More so, high Procr expression was also detected and used as indicator for subsets of triple-negative breast cancers (TNBCs). Previous study has revealed Procr as a target of Wnt/β-catenin signaling; however, direct upstream regulatory mechanism of Procr remains unknown. To comprehend the molecular role of Procr during physiology and pathology, elucidating the upstream effectors of Procr is necessary. Here, we provide a system for screening negative regulators of Procr, which could be adapted for broad molecular analysis on membrane proteins. Results: We established a screening system which combines CRISPR-Cas9 guided gene disruption with fluorescence activated cell sorting technique (FACS). CommaDβ (murine epithelial cells line) was used for the initial Procr upstream effector screening using lentiviral CRISPR-gRNA library. Shortlisted genes were further validated through individual lentiviral gRNA infection followed by Procr expression evaluation. Adam17 was identified as a specific negative inhibitor of Procr expression. In addition, MDA-MB-231 cells and Hs578T cells (human breast cancer cell lines) were used to verify the conserved regulation of ADAM17 over PROCR expression. Conclusion: We established an efficient CRISPR-Cas9/FACS screening system, which identifies the regulators of membrane proteins. Through this system, we identified Adam17 as the negative regulator of Procr membrane expression both in mammary epithelial cells and breast cancer cells.</p
