Faten Zaibak’s research while affiliated with The Royal Children's Hospital and other places

Multipotent mesenchymal stromal cells derived from human placenta (pMSCs), and unrestricted somatic stem cells (USSCs) derived from cord blood share many properties with human bone marrow-derived mesenchymal stromal cells (bmMSCs) and are currently in clinical trials for a wide range of clinical settings. Here we present gene expression profiles of human cord blood-derived unrestricted somatic stem cells (USSCs), human placental-derived mesenchymal stem cells (hpMSCs), and human bone marrow-derived mesenchymal stromal cells (bmMSCs), all derived from four different donors. The microarray data are available on the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-TABM-880. Additionally, the data has been integrated into a public portal, www.stemformatics.org. Our data provide a resource for understanding the differences in MSCs derived from different tissues.

Activin receptor (ACVR) protein levels in USSC grown in serum free conditions. Representative light micrographs depicting the localization of ACVR (brown staining) in USSC grown in serum free conditions (SCPM containing 0% FCS; 400×). Nuclei are counterstained blue with Haematoxylin. USSC 1 (left column) and USSC 2 (right column) expressed ACVR1b (A, B), ACVR2a (C, D) and ACVR2b (E, F). No staining was detected in the IgG controls (G, H).

Activin A induction strategy. Cells were divided into one of three treatment groups; Normal serum control, Low serum control and Activin A. Normal serum control cells were seeded in SCPM containing 30% FCS and then cultured in SCPM containing 30% FCS for three days. For all treatment groups, the media was changed daily. Low serum control and Activin A cells were seeded in serum-free SCPM. Media was replaced with the following; serum-free SCPM on Day 1, SCPM containing 0.2% FCS on Day 2 and SCPM containing 2% FCS on Day 3; the media for Activin A cells on Days 1 to 3 was supplemented with 100 ng/ml Activin A.

Cell proliferation index. The number of cells counted when USSC grown in SCPM containing 0%, 1%, 2% or 30% FCS for seven days are expressed as a percentage of the number of cells seeded. <100% initial seeding density indicates cell death has occurred, while >100% initial seeding density indicates cell proliferation has occurred. Different letters denote statistical significance (P < 0.05).

Unrestricted somatic stem cells (USSC) derived from umbilical cord blood are an attractive alternative to human embryonic stem cells (hESC) for cellular therapy. USSC are capable of forming cells representative of all three germ line layers. The aim of this study was to determine the potential of USSC to form definitive endoderm following induction with Activin A, a protein known to specify definitive endoderm formation of hESC. USSC were cultured for (1) three days with or without 100 ng/ml Activin A in either serum-free, low-serum or serum-containing media, (2) three days with or without 100 ng/ml Activin A in combination with 10 ng/ml FGF4 in pre-induction medium, or (3) four days with or without small molecules Induce Definitive Endoderm (IDE1, 100 nM; IDE2, 200 nM) in serum-free media. Formation of definitive endoderm was assessed using RT-PCR for gene markers of endoderm (Sox17, FOXA2 and TTF1) and lung epithelium (surfactant protein C; SPC) and cystic fibrosis transmembrane conductance regulator; CFTR). The differentiation capacity of Activin A treated USSC was also assessed. Activin A or IDE1/2 induced formation of Sox17+ definitive endoderm from hESC but not from USSC. Activin A treated USSC retained their capacity to form cells of the ectoderm (nerve), mesoderm (bone) and endoderm (lung). Activin A in combination with FGF4 did not induce formation of Sox17+ definitive endoderm from USSC. USSC express both Activin A receptor subunits at the mRNA and protein level, indicating that these cells are capable of binding Activin A. Stimulation of the Nodal signaling pathway with Activin A or IDE1/2 is insufficient to induce definitive endoderm formation from USSC, indicating that USSC differ in their stem cell potential from hESC.

Human umbilical cord blood-derived unrestricted somatic stem cells (USSCs), which are capable of multilineage differentiation, are currently under investigation for a number of therapeutic applications. A major obstacle to their clinical use is the fact that in vitro expansion is still dependent upon fetal calf serum, which could be a source of pathogens. In this study, we investigate the capacity of three different stem cell culture media to support USSCs in serum-free conditions; HEScGRO, PSM and USSC growth medium ACF. Our findings demonstrate that USSCs do not grow in HEScGRO or PSM, but we were able to isolate, proliferate and maintain multipotency of three USSC lines in USSC growth medium ACF. For the first one to three passages, cells grown in USSC growth medium ACF proliferate and maintain their morphology, but with continued passaging the cells form spherical cell aggregates. Upon dissociation of spheres, cells continue to grow in suspension and form new spheres. Dissociated cells can also revert to monolayer growth when cultured on extracellular matrix support (fibronectin or gelatin), or in medium containing fetal calf serum. Analysis of markers associated with pluripotency (Oct4 and Sox2) and differentiation (FoxA2, Brachyury, Goosecoid, Nestin, Pax6, Gata6 and Cytokeratin 8) confirms that cells in the spheres maintain their gene expression profile. The cells in the spheres also retain the ability to differentiate in vitro to form cells representative of the three germline layers after five passages. These data suggest that USSC growth medium ACF maintains USSCs in an undifferentiated state and supports growth in suspension. This is the first demonstration that USSCs can grow in a serum- and animal component-free medium and that USSCs can form spheres.

Stem cells from a patient with a genetic disease could be used for cell therapy if it were possible to insert a functional copy of the defective gene. In this study, we investigate the transfection and subsequent integration of large genomic fragments into human cord blood-derived multipotent stem cells. We describe for the first time the creation of clonal stem cells carrying a human bacterial artificial chromosome (BAC) containing the Friedreich ataxia locus with an enhanced green fluorescent protein (EGFP) reporter gene fused to exon 5a of the frataxin (FXN) gene. Integration of the BAC into the host cell genome was confirmed by PCR, Southern blot and fluorescent in situ hybridization analysis. Reverse transcription-PCR and flow cytometry confirmed expression of FXN-EGFP. Correct mitochondrial localization of the protein was confirmed using fluorescent microscopy. The transfected stem cells also retained the ability to differentiate into cells from all three germline layers, as demonstrated by the capacity to form neuron-specific beta-tubulin-expressing cells, Alizarin Red S-positive bone-like cells, and epithelial-like cells expressing surfactant protein C. This is the first study to demonstrate that cord blood-derived multipotent stem cells may be useful targets for gene therapy applications using large genomic loci.