In Vitro Differentiation Of The HFⅧ-integrated HESCs To Mesenchymal Stem Cells

Hemophilia A is one of the highlights in gene therapy study. HA is a single gene disorder caused by FVIII gene lack of function. In previous study, we have successfully targeted the reconstructive human coagulation factor VIII (hFVIII) gene, hFVIII-BDDAK39, into the rDNA locus of hES cell line H9(named ET5) using a human rDNA targeting vector (pHrneo) carrying hFVIII-BDDAK39. We will differentiate ET5into specific tissue cells for transplantation of HA. Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into a variety of cell types. In vivo, autologous or heterogeneous MSC transplantation has demonstrated therapeutic efficacy in treating diseases or repairing damaged tissues.MSCs also have a negligible risk of teratoma formation as well as immunosuppressive features.These characteristics of MSCs make them well-suited as gene delivery vehicles. However, the availability of tissues for their isolation remains limiting and requires invasive procedures. Ex vivo expansion of MSCs, although significant, is nonetheless finite. In this study, we will induce in vitro differentiation of ET5into MSC and analyze expression of tansgene hFVIII in MSCs derived from ET5. Our results may provide the experimental basis for the future use in gene therapy for hemophilia A. Objective:To establish the technology of in vitro differentiation of hESCs into MSCs, study the characteristics between the hESCs-derived MSCs and BM-derived MSCs, and analyze the expression of transgene hFVIII in MSCs derived from the hESCs with hFVIII site-specific integration.Methods:1. After AKP staining of ET5, bFGF and PDGF-BB were added into medium to induce in vitro differentiation of ET5into MSCs. Karyotype analysis was performed in the long-term cultured ET5-derived MSCs.2. ET5-derived MSCs were in vitro differentiated into adipocytes, chondrocytes, and osteocytes.3. Expression of surface markers was analyzed by flow cytometry in ET5, ET5-derived MSCs and BM-MSCs.4. Expression of the marker genes (Oct4, Sox2and Nanog) was characterized by semiquantitative RT-PCR in ET5-derived MSCs, BM-MSCs and ET5.5. Expression of tansgene hFVIII in ET5-derived MSCs was determined using RT-PCR.Results:1. AKP staining are positive in ET5, but negative in ET5-derived MSCs. The karyotype is keeping normal during long term passaging and culture of the ET5-derived MSCs.2. The ET5-derived MSCs were validated to maintain the capacity of in vitro differentiatiton into adipocytes, chondrocytes, and osteocytes.3. Expression of surface markers in BM-MSCs was consistent with ET5-MSCs.4. Expression of OCT4, SOX2and Nanog were downregulated during the differentiation from hESCs into MSCs.5. Expression of the tansgene hFVIII at RNA level was detected.Conclusion:We have established the technology of in vitro differentiation of hESCs into MSCs with high efficiency, by which the characteristics of hESC-derived MSCs were similar to those of BM-derived MSCs. Expression of the transgene hFVIII could be detected in MSCs derived from the hESCs with hFVIII site-specific integration.