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

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.

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

Prenatal transplantation of human amniotic fluid stem cell could improve clinical outcome of type III spinal muscular atrophy in mice

Spinal muscular atrophy (SMA) is a single gene disorder affecting motor function in uterus. Amniotic fluid is an alternative source of stem cell to ameliorate SMA. Therefore, this study aims to examine the therapeutic potential of Human amniotic fluid stem cell (hAFSC) for SMA. Our SMA model mice were generated by deletion of exon 7 of Smn gene and knock-in of human SMN2. A total of 16 SMA model mice were injected with 1 × 105 hAFSC in uterus, and the other 16 mice served as the negative control. Motor function was analyzed by three behavioral tests. Engraftment of hAFSC in organs were assessed by flow cytometry and RNA scope. Frequency of myocytes, neurons and innervated receptors were estimated by staining. With hAFSC transplantation, 15 fetuses survived (93.75% survival) and showed better performance in all motor function tests. Higher engraftment frequency were observed in muscle and liver. Besides, the muscle with hAFSC transplantation expressed much laminin α and PAX-7. Significantly higher frequency of myocytes, neurons and innervated receptors were observed. In our study, hAFSC engrafted on neuromuscular organs and improved cellular and behavioral outcomes of SMA model mice. This fetal therapy could preserve the time window and treat in the uterus.

Amniotic Fluid Stem Cell-Derived Extracellular Vesicles Counteract Steroid-Induced Osteoporosis In Vitro

Background—Osteoporosis is characterized by defects in both quality and quantity of bone tissue, which imply high susceptibility to fractures with limitations of autonomy. Current therapies for osteoporosis are mostly concentrated on how to inhibit bone resorption but give serious adverse effects. Therefore, more effective and safer therapies are needed that even encourage bone formation. Here we examined the effect of extracellular vesicles secreted by human amniotic fluid stem cells (AFSC) (AFSC-EV) on a model of osteoporosis in vitro. Methods—human AFSC-EV were added to the culture medium of a human pre-osteoblast cell line (HOB) induced to differentiate, and then treated with dexamethasone as osteoporosis inducer. Aspects of differentiation and viability were assessed by immunofluorescence, Western blot, mass spectrometry, and histological assays. Since steroids induce oxidative stress, the levels of reactive oxygen species and of redox related proteins were evaluated. Results—AFSC-EV were able to ameliorate the differentiation ability of HOB both in the case of pre-osteoblasts and when the differentiation process was affected by dexamethasone. Moreover, the viability was increased and parallelly apoptotic markers were reduced. The presence of EV positively modulated the redox unbalance due to dexamethasone. Conclusion—these findings demonstrated that EV from hAFSC have the ability to recover precursor cell potential and delay local bone loss in steroid-related osteoporosis.

Prenatal Transplantation of Human Amniotic Fluid Stem Cell Could Improve Clinical Outcome of Type III Spinal Muscular Atrophy

BackgroundType III spinal muscular atrophy (SMA) is a single gene disorder affecting motor function in uterus. Several types of stem cells were utilized to ameliorate SMA based on its capability of regeneration and differentiation. Amniotic fluid is an alternative source of stem cells and is safely sampled without ethical issues. Human amniotic fluid stem cell (hAFSC) shared common surface markers of mesenchymal stem cell. Therefore, this study aims to examine the therapeutic potential of hAFSC for SMA. MethodsOur SMA model mice were generated by deletion of exon 7 of Smn gene and knock-in of human SMN2. A total of 16 SMA model mice were injected with 1x105 hAFSC in uterus, and the other 16 mice served as the negative control. Motor function was analyzed by Rotarod maintenance test, tilting test and grasping test every two months. Twelve months after transplantation, all organs were extracted for post-mortem analysis. Engraftment of hAFSC in organs were assessed by flow cytometry and RNA scope. To observe the function of neuromuscular junction, frequency of myocytes, neurons and innervated receptors were estimated by H&E, methylene blue and immunocytochemistry staining. ResultsWith hAFSC transplantation, 15 fetuses from 5 dams survived (15 of 16, 93.75% survival) and showed better performance in all three motor function tests. Higher engraftment frequency in organs were observed in muscle and liver after hAFSC transplantation. Besides, the muscle of SMA mice with hAFSC transplantation expressed much laminin α and PAX-7. Significantly higher frequency of myocytes, neurons and innervated receptors were observed after hAFSC transplantation. ConclusionsIn our study, hAFSC engrafted on neuromuscular organs and improved cellular and behavioral outcomes of SMA model mice. This fetal therapy could preserve the time window and treat in the uterus to avoid irreversible damage.