Protein and molecular characterization of a clinically compliant amniotic fluid stem cell derived extracellular vesicle fraction capable of accelerating muscle regeneration through the enhancement of angiogenesis

The secretome of human amniotic fluid stem cells (AFSC) has great potential as a therapeutic agent in regenerative medicine. However it must be produced in a clinically compliant manner before it can be used in humans. Here we developed a means of producing a biologically active secretome from AFSCs that is free of all exogenous molecules. We demonstrate that the full secretome is capable of promoting stem cell proliferation, migration and protection of cells against senescence. Furthermore, it has significant anti-inflammatory properties. Most importantly we show that it promotes tissue regeneration in a model of muscle damage. We then demonstrate that the secretome contains extracellular vesicles (EV) that harbour much but not all the biological activity of the whole secretome. Proteomic characterisation of the EV and free secretome fraction show the presence of numerous molecules specific to each fraction that could be key regulators of tissue regeneration. Intriguingly we show that the EVs only contain miRNA and not mRNA. This suggests tissue regeneration in the host is mediated by the action of EVs modifying existing, rather than the imposition of new, signalling pathways. The EVs harbour significant anti-inflammatory activity as well as promoting angiogenesis; the latter may be the mechanistic explanation for their ability to promote muscle regeneration after cardiotoxin injury

In Utero Gene Therapy (IUGT) Using GLOBE Lentiviral Vector Phenotypically Corrects the Heterozygous Humanised Mouse Model and Its Progress Can Be Monitored Using MRI Techniques

Funder: UK Thalassaemia SocietyAbstract: In utero gene therapy (IUGT) to the fetal hematopoietic compartment could be used to treat congenital blood disorders such as β-thalassemia. A humanised mouse model of β-thalassemia was used, in which heterozygous animals are anaemic with splenomegaly and extramedullary hematopoiesis. Intrahepatic in utero injections of a β globin-expressing lentiviral vector (GLOBE), were performed in fetuses at E13.5 of gestation. We analysed animals at 12 and 32 weeks of age, for vector copy number in bone marrow, peripheral blood liver and spleen and we performed integration site analysis. Compared to noninjected heterozygous animals IUGT normalised blood haemoglobin levels and spleen weight. Integration site analysis showed polyclonality. The left ventricular ejection fraction measured using magnetic resonance imaging (MRI) in treated heterozygous animals was similar to that of normal non-β-thalassemic mice but significantly higher than untreated heterozygous thalassemia mice suggesting that IUGT ameliorated poor cardiac function. GLOBE LV-mediated IUGT normalised the haematological and anatomical phenotype in a heterozygous humanised model of β-thalassemia

Publisher Correction: In Utero Gene Therapy (IUGT) Using GLOBE Lentiviral Vector Phenotypically Corrects the Heterozygous Humanised Mouse Model and Its Progress Can Be Monitored Using MRI Techniques

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Publisher Correction: In Utero Gene Therapy (IUGT) Using GLOBE Lentiviral Vector Phenotypically Corrects the Heterozygous Humanised Mouse Model and Its Progress Can Be Monitored Using MRI Techniques.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Long-Term Hematopoietic Engraftment of Congenic Amniotic Fluid Stem Cells after in Utero Intraperitoneal Transplantation to Immune Competent Mice

Clinical success of in utero transplantation (IUT) using allogeneic hematopoietic stem cells (HSCs) has been limited to fetuses that lack an immune response to allogeneic cells due to severe immunological defects, and where transplanted genetically normal cells have a proliferative or survival advantage. Amniotic fluid (AF) is an autologous source of stem cells with hematopoietic potential that could be used to treat congenital blood disorders. We compared the ability of congenic and allogeneic mouse AF stem cells (AFSC) to engraft the hematopoietic system of time-mated C57BL/6J mice (E13.5). At 4 and 16 weeks of age, multilineage donor engraftment was higher in congenic versus allogeneic animals. In vitro mixed lymphocyte reaction confirmed an immune response in the allogeneic group with higher CD4 and CD8 cell counts and increased proliferation of stimulated lymphocytes. IUT with congenic cells resulted in 100% of donor animals having chimerism of around 8% and successful hematopoietic long-term engraftment in immune-competent mice when compared with IUT with allogeneic cells. AFSCs may be useful for autologous cell/gene therapy approaches in fetuses diagnosed with congenital hematopoietic disorders

In utero therapy for congenital disorders using amniotic fluid stem cells.

Congenital diseases are responsible for over a third of all pediatric hospital admissions. Advances in prenatal screening and molecular diagnosis have allowed the detection of many life-threatening genetic diseases early in gestation. In utero transplantation with stem cells (IUT) could cure affected fetuses but so far in humans, successful IUT using allogeneic haematopoietic stem cells (HSCs), has been limited to fetuses with severe immunologic defects and more recently IUT with allogeneic mesenchymal stem cell transplantation, has improved phenotype in osteogenesis imperfecta. The option of preemptive treatment of congenital diseases in utero by stem cell or gene therapy are encouraging as it changes the perspective of congenital diseases. Thus, avoiding the need for post-natal treatment and reducing future costs. AFS have been isolated and characterized in human, mice, rodents, rabbit and sheep and can be a potential source of cells for therapeutic applications in a multitude of disorders that can be treated prenatally or postnatally. These cells have demonstrated the potential of repair in a range of disease models such as neurological disorder, tracheal repair, bladder injury and diaphragmatic hernia repair in adult or neonate stage. Several groups have shown the use of AFS in in utero therapy in rodents as well as sheep models. These results have been encouraging, thus allowing us to continue with the research and optimizing the procedures and experiments so as to allow it to be translated into clinic

In Utero Gene Therapy (IUGT) Using GLOBE Lentiviral Vector Phenotypically Corrects the Heterozygous Humanised Mouse Model and Its Progress Can Be Monitored Using MRI Techniques

Funder: UK Thalassaemia SocietyAbstract: In utero gene therapy (IUGT) to the fetal hematopoietic compartment could be used to treat congenital blood disorders such as β-thalassemia. A humanised mouse model of β-thalassemia was used, in which heterozygous animals are anaemic with splenomegaly and extramedullary hematopoiesis. Intrahepatic in utero injections of a β globin-expressing lentiviral vector (GLOBE), were performed in fetuses at E13.5 of gestation. We analysed animals at 12 and 32 weeks of age, for vector copy number in bone marrow, peripheral blood liver and spleen and we performed integration site analysis. Compared to noninjected heterozygous animals IUGT normalised blood haemoglobin levels and spleen weight. Integration site analysis showed polyclonality. The left ventricular ejection fraction measured using magnetic resonance imaging (MRI) in treated heterozygous animals was similar to that of normal non-β-thalassemic mice but significantly higher than untreated heterozygous thalassemia mice suggesting that IUGT ameliorated poor cardiac function. GLOBE LV-mediated IUGT normalised the haematological and anatomical phenotype in a heterozygous humanised model of β-thalassemia