Durrgah L. Ramachandra

Microgel Iron Oxide Nanoparticles for Tracking Human Fetal Mesenchymal Stem Cells Through Magnetic Resonance Imaging

Stem cell transplantation for regenerative medicine has made significant progress in various injury models, with the development of modalities to track stem cell fate and migration post‐transplantation being currently pursued rigorously. Magnetic resonance imaging (MRI) allows serial high‐resolution in vivo detection of transplanted stem cells labeled with iron oxide particles, but has been hampered by low labeling efficiencies. Here, we describe the use of microgel iron oxide (MGIO) particles of diameters spanning 100‐750 nm for labeling human fetal mesenchymal stem cells (hfMSCs) for MRI tracking. We found that MGIO particle uptake by hfMSCs was size dependent, with 600‐nm MGIO (M600) particles demonstrating three‐ to sixfold higher iron loading than the clinical particle ferucarbotran (33‐263 versus 9.6‐42.0 pg iron/hfMSC; p < .001). Cell labeling with either M600 particles or ferucarbotran did not affect either cellular proliferation or trilineage differentiation into osteoblasts, adipocytes, and chondrocytes, despite differences in gene expression on a genome‐wide microarray analysis. Cell tracking in a rat photothrombotic stroke model using a clinical 1.5‐T MRI scanner demonstrated the migration of labeled hfMSCs from the contralateral cortex to the stroke injury, with M600 particles achieving a five‐ to sevenfold higher sensitivity for MRI detection than ferucarbotran (p < .05). However, model‐related cellular necrosis and acute inflammation limited the survival of hfMSCs beyond 5‐12 days. The use of M600 particles allowed high detection sensitivity with low cellular toxicity to be achieved through a simple incubation protocol, and may thus be useful for cellular tracking using standard clinical MRI scanners. STEM CELLS 2009;27:1921–1931

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 (IUT) with stem cells could cure affected fetuses but so far in humans, successful IUT using allogeneic hematopoietic 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 options of preemptive treatment of congenital diseases in utero by stem cell or gene therapy changes the perspective of congenital diseases since it may avoid the need for postnatal treatment and reduce future costs. Amniotic fluid stem (AFS) cells have been isolated and characterized in human, mice, rodents, rabbit, and sheep and are a potential source of cells for therapeutic applications in disorders for treatment prenatally or postnatally. Gene transfer to the cells with long-term transgenic protein expression is feasible. Recently, pre-clinical autologous transplantation of transduced cells has been achieved in fetal sheep using minimally invasive ultrasound guided injection techniques. Clinically relevant levels of transgenic protein were expressed in the blood of transplanted lambs for at least 6 months. The cells have also demonstrated the potential of repair in a range of pre-clinical disease models such as neurological disorders, tracheal repair, bladder injury, and diaphragmatic hernia repair in neonates or adults. These results have been encouraging, and bring personalized tissue engineering for prenatal treatment of genetic disorders closer to the clinic.

Sheep CD34+ Amniotic Fluid Cells Have Hematopoietic Potential and Engraft After Autologous In Utero Transplantation

Unmatched allogeneic in utero stem cell transplantation (IUSCT) produces poor engraftment unless the fetus has congenital immunodeficiency, probably because of maternal and fetal immune responses to injected cells. We studied the functional hematopoietic potential of transduced green fluorescent protein (GFP+) sheep amniotic fluid (AF) stem cells, before and after autologous IUSCT. CD34+ cells were selected from first trimester sheep AF, transduced overnight, and injected intravenously into NOD‐SCID‐gamma (NSG) mice. At 3 months, primary recipient bone marrow (BM) was injected into secondary NSG recipients. GFP+ cells were detected in the hematopoietic organs and peripheral blood of primary and secondary recipients at 3 months. Autologous IUSCT (transduced GFP+CD34+AF) was performed in fetal sheep. Six months postnatally, lamb BM was injected into secondary NSG recipients. GFP+ cells were detected in the peripheral blood of primary and secondary recipients. This confirms the hematopoietic potential of AF stem cells supporting the concept of autologous IUSCT to treat congenital hematopoietic disease. Stem Cells 2015;33:122–132

Protein and Molecular Characterization of a Clinically Compliant Amniotic Fluid Stem Cell-Derived Extracellular Vesicle Fraction Capable of Accelerating Muscle Regeneration Through Enhancement of Angiogenesis

The secretome of human amniotic fluid stem cells (AFSCs) 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. In this study, 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 (EVs) that harbor much, but not all, of the biological activity of the whole secretome. Proteomic characterization of the EV and free secretome fraction shows 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 that tissue regeneration in the host is mediated by the action of EVs modifying existing, rather than imposing new, signaling pathways. The EVs harbor significant anti-inflammatory activity as well as promote angiogenesis, the latter may be the mechanistic explanation for their ability to promote muscle regeneration after cardiotoxin injury.