An optimized procedure for preparation of conditioned medium from Wharton's jelly mesenchymal stromal cells isolated from umbilical cord.

Cell-free therapy based on conditioned medium derived from mesenchymal stromal cells (MSCs) has gained attention in the field of protective and regenerative medicine. However, the exact composition and properties of MSC-derived conditioned media can vary greatly depending on multiple parameters, which hamper standardization. In this study, we have optimized a procedure for preparation of conditioned medium starting from efficient isolation, propagation and characterization of MSCs from human umbilical cord, using a culture medium supplemented with human platelet lysate as an alternative source to fetal bovine serum. Our procedure successfully maximizes the yield of viable MSCs that maintain canonical key features. Importantly, under these conditions, the compositional profile and biological effects elicited by the conditioned medium preparations derived from these MSC populations do not depend on donor individuality. Moreover, approximately 120 L of conditioned medium could be obtained from a single umbilical cord, which provides a suitable framework to produce industrial amounts of toxic-free conditioned medium with predictable composition.

Non-Primate Lentiviral Vectors and Their Applications in Gene Therapy for Ocular Disorders.

Lentiviruses have a number of molecular features in common, starting with the ability to integrate their genetic material into the genome of non-dividing infected cells. A peculiar property of non-primate lentiviruses consists in their incapability to infect and induce diseases in humans, thus providing the main rationale for deriving biologically safe lentiviral vectors for gene therapy applications. In this review, we first give an overview of non-primate lentiviruses, highlighting their common and distinctive molecular characteristics together with key concepts in the molecular biology of lentiviruses. We next examine the bioengineering strategies leading to the conversion of lentiviruses into recombinant lentiviral vectors, discussing their potential clinical applications in ophthalmological research. Finally, we highlight the invaluable role of animal organisms, including the emerging zebrafish model, in ocular gene therapy based on non-primate lentiviral vectors and in ophthalmology research and vision science in general.

Wharton's Jelly Mesenchymal Stromal Cells Support the Expansion of Cord Blood-derived CD34+ Cells Mimicking a Hematopoietic Niche in a Direct Cell-cell Contact Culture System.

Wharton's jelly mesenchymal stromal cells (WJ-MSCs) have been recently exploited as a feeder layer in coculture systems to expand umbilical cord blood-hematopoietic stem/progenitor cells (UCB-HSPCs). Here, we investigated the role of WJ-MSCs in supporting ex vivo UCB-HSPC expansion either when cultured in direct contact (DC) with WJ-MSCs or separated by a transwell system or in the presence of WJ-MSC-conditioned medium. We found, in short-term culture, a greater degree of expansion of UCB-CD34+ cells in a DC system (15.7 ± 4.1-fold increase) with respect to the other conditions. Moreover, in DC, we evidenced two different CD34+ cell populations (one floating and one adherent to WJ-MSCs) with different phenotypic and functional characteristics. Both multipotent CD34+/CD38- and lineage-committed CD34+/CD38+ hematopoietic progenitors were expanded in a DC system. The former were significantly more represented in the adherent cell fraction than in the floating one (18.7 ± 11.2% vs. 9.7 ± 7.9% over the total CD34+ cells). Short-term colony forming unit (CFU) assays showed that HSPCs adherent to the stromal layer were able to generate a higher frequency of immature colonies (CFU-granulocyte/macrophage and burst-forming unit erythroid/large colonies) with respect to the floating cells. In the attempt to identify molecules that may play a role in supporting the observed ex vivo HSPC growth, we performed secretome analyses. We found a number of proteins involved in the HSPC homing, self-renewal, and differentiation in all tested conditions. It is important to note that a set of sixteen proteins, which are only in part reported to be expressed in any hematopoietic niche, were exclusively found in the DC system secretome. In conclusion, WJ-MSCs allowed a significant ex vivo expansion of multipotent as well as committed HSPCs. This may be relevant for future clinical applications.

Wharton's Jelly Mesenchymal Stromal Cells as a Feeder Layer for the Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells: a Review.

In recent years, umbilical cord blood (UCB) has been widely used as an alternative source to bone marrow (BM) for transplantation of hematopoietic stem and progenitor cells (HSPCs) in a variety of hematological and non-hematological disorders. Nevertheless, the insufficient number of UCB-HSPCs for graft represents a major challenge. HSPCs ex vivo expansion prior to transplantation is a valid strategy to overcome this limit. Several attempts to optimize the expansion conditions have been reported, including the use of mesenchymal stromal cells (MSCs) as feeder layer. Wharton's Jelly (WJ), the main component of umbilical cord (UC) matrix, is especially rich in MSCs, which are considered ideal candidates for feeder layer in co-culture systems. In fact, they can be easily harvested and grow robustly in culture, producing a confluent monolayer in a short time. Similarly to bone marrow-mesenchymal stromal cells (BM-MSCs), WJ-derived MSCs (WJ-MSCs) have been used to support hematopoiesis in vitro and in vivo. Here, we review the rationale for using MSCs, particularly WJ-MSCs, as a feeder layer for UCB-HSPCs ex vivo expansion. In addition, we report the main findings attesting the use of these MSCs as a support in hematopoiesis.

The Sea Urchin sns5 Chromatin Insulator Shapes the Chromatin Architecture of a Lentivirus Vector Integrated in the Mammalian Genome.

Lentivirus vectors are presently the favorite vehicles for therapeutic gene transfer in hematopoietic cells. Nonetheless, these vectors integrate randomly throughout the genome, exhibiting variegation of transgene expression due to the spreading of heterochromatin into the vector sequences. Moreover, the cis-regulatory elements harbored by the vector could disturb the proper transcription of resident genes neighboring the integration site. The incorporation of chromatin insulators in flanking position to the transferred unit can alleviate both the above-mentioned dangerous effects, due to the insulator-specific barrier and enhancer-blocking activities. In this study, we report the valuable properties of the sea urchin-derived sns5 insulator in improving the expression efficiency of a lentivirus vector integrated in the mammalian erythroid genome. We show that these results neither reflect an intrinsic sns5 enhancer activity nor rely on the recruitment of the erythroid-specific GATA-1 factor to sns5. Furthermore, by using the Chromosome Conformation Capture technology, we report that a single copy of the sns5-insulated vector is specifically organized into an independent chromatin loop at the provirus locus. Our results not only provide new clues concerning the molecular mechanism of sns5 function in the erythroid genome but also reassure the use of sns5 to improve the performance of gene therapy vectors.

The sea urchin sns5 insulator protects retroviral vectors from chromosomal position effects by maintaining active chromatin structure.

Silencing and position-effect (PE) variegation (PEV), which is due to integration of viral vectors in heterochromatin regions, are considered significant obstacles to obtaining a consistent level of transgene expression in gene therapy. The inclusion of chromatin insulators into vectors has been proposed to counteract this position-dependent variegation of transgene expression. Here, we show that the sea urchin chromatin insulator, sns5, protects a recombinant gamma-retroviral vector from the negative influence of chromatin in erythroid milieu. This element increases the probability of vector expression at different chromosomal integration sites, which reduces both silencing and PEV. By chromatin immunoprecipitation (ChIP) analysis, we demonstrated the specific binding of GATA1 and OCT1 transcription factors and the enrichment of hyperacetylated nucleosomes to sns5 sequences. The results suggest that this new insulator is able to maintain a euchromatin state inside the provirus locus with mechanisms that are common to other characterized insulators. On the basis of its ability to function as barrier element in erythroid milieu and to bind the erythroid specific factor GATA1, the inclusion of sns5 insulator in viral vectors may be of practical benefit in gene transfer applications and, in particular, for gene therapy of erythroid disorders.

Functional characterization of the sea urchin sns chromatin insulator in erythroid cells.

Chromatin insulators are regulatory elements that determine domains of genetic functions. We have previously described the characterization of a 265 bp insulator element, termed sns, localized at the 3' end of the early histone H2A gene of the sea urchin Paracentrotus lividus. This sequence contains three cis-acting elements (Box A, Box B, and Box C + T) all needed for the enhancer-blocking activity in both sea urchin and human cells. The goal of this study was to further characterize the sea urchin sns insulator in the erythroid environment. We employed colony assays in human (K562) and mouse (MEL) erythroid cell lines. We tested the capability of sns to interfere with the communication between the 5'HS2 enhancer of the human beta-globin LCR and the gamma-globin promoter. We found that the sns sequence displays directional enhancer-blocking activity. By the use of antibodies against known DNA binding proteins, in electrophoretic mobility shift assays, we demonstrated the binding of the erythroid-specific GATA-1 and the ubiquitous Oct-1 and Sp1 transcription factors. These factors bind to Box A, Box B, and Box C + T, respectively, in both K562 and MEL nuclear extracts. These results may have significant implications for the conservation of insulator function in evolutionary distant organisms and may prove to be of practical benefit in gene transfer applications for erythroid disorders such as hemoglobinopathies and thalassemias.