Identification of Nephron Progenitor Cells from WT1 through Amniotic Fluid as a Stem Cell Initiator

Background: The incidence of kidney disease is prevailing worldwide and there is an urgent requirement for regenerative techniques such as stem cells. Objective: To identify nephron progenitor cells from transcriptional factor WT1. Study Design: Experimental analytical study Place and Duration of Study: Dow Research Institute of Biotechnology & Biomedical Sciences, Dow International Medical College, Karachi from 1st January 2019 to 31stDecember 2019 Methodology: 40 ml of amniotic fluid was extracted from 10 full term women at the time of elective caesarean. Using cell culturing, inverted phase contrast microscopy and flow cytometry techniques in addition to immune-florescence the nephron progenitor cells were identified. Results: The mean age of women was 30.3±0.4 years. Out of total 10 million WT1 expressed in three samples 1.4 million nephron progenitor cells were identified. Conclusion: Identification of nephron progenitor cells is feasible procedure for designing stem cell lines through amniotic fluid. Key words: Progenitor cell, Amniotic fluid, Stem cell initiator

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Autophagy is impaired in fetal hypoplastic lungs and rescued by administration of amniotic fluid stem cell extracellular vesicles

10.1101/2022.01.12.476078 ◽

2022 ◽

Author(s):

Kasra Khalaj ◽

Lina Antounians ◽

Rebeca Lopes Figueira ◽

Martin Post ◽

Augusto Zani

Keyword(s):

Stem Cell ◽

Amniotic Fluid ◽

Extracellular Vesicles ◽

Pulmonary Hypoplasia ◽

Fetal Lung ◽

Branching Morphogenesis ◽

Lung Epithelial Cells ◽

Normal Lung ◽

Amniotic Fluid Stem Cell ◽

Human Fetal Lung

Rationale: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by reduced branching morphogenesis, which is responsible for poor clinical outcomes. Administration of amniotic fluid stem cell extracellular vesicles (AFSC-EVs) rescues branching morphogenesis in rodent fetal models of pulmonary hypoplasia. Herein, we hypothesized that AFSC-EVs exert their regenerative potential by affecting autophagy, a process required for normal lung development. Objectives: To evaluate autophagy in hypoplastic lungs throughout gestation and establish whether AFSC-EV administration improves branching morphogenesis through autophagy-mediated mechanisms. Methods: EVs were isolated from c-kit+ AFSC conditioned medium by ultracentrifugation and characterized for size, morphology, and EV markers. Branching morphogenesis was inhibited in rat fetuses by nitrofen administration to dams and in human fetal lung explants by blocking RAC1 activity with NSC23766. Expression of autophagy activators (BECN1 and ATG5) and adaptor (SQSTM1/p62) was analyzed in vitro (rat and human fetal lung explants) and in vivo (rat fetal lungs). Mechanistic studies on rat fetal primary lung epithelial cells were conducted using inhibitors for microRNA-17 and -20a contained in the AFSC-EV cargo and known to regulate autophagy. Measurements and Main Results: Rat and human models of fetal pulmonary hypoplasia showed reduced autophagy mainly at pseudoglandular and canalicular stages. AFSC-EV administration restored autophagy in both pulmonary hypoplasia models by transferring miR-17~92 cluster members contained in the EV cargo. Conclusions: AFSC-EV treatment rescues branching morphogenesis partly by restoring autophagy through miRNA cargo transfer. This study enhances our understanding of pulmonary hypoplasia pathogenesis and creates new opportunities for fetal therapeutic intervention in CDH babies.

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