Comparison of different gene addition strategies to modify placental derived-mesenchymal stromal cells to produce FVIII.

Introduction: Placenta-derived mesenchymal cells (PLCs) endogenously produce FVIII, which makes them ideally suited for cell-based fVIII gene delivery. We have previously reported that human PLCs can be efficiently modified with a lentiviral vector encoding a bioengineered, expression/secretion-optimized fVIII transgene (ET3) and durably produce clinically relevant levels of functionally active FVIII. The objective of the present study was to investigate whether CRISPR/Cas9 can be used to achieve location-specific insertion of a fVIII transgene into a genomic safe harbor, thereby eliminating the potential risks arising from the semi-random genomic integration inherent to lentiviral vectors. We hypothesized this approach would improve the safety of the PLC-based gene delivery platform and might also enhance the therapeutic effect by eliminating chromatin-related transgene silencing. Methods: We used CRISPR/Cas9 to attempt to insert the bioengineered fVIII transgene "lcoET3" into the AAVS1 site of PLCs (CRISPR-lcoET3) and determined their subsequent levels of FVIII production, comparing results with this approach to those achieved using lentivector transduction (LV-lcoET3) and plasmid transfection (Plasmid-lcoET3). In addition, since liver-derived sinusoidal endothelial cells (LSECs) are the native site of FVIII production in the body, we also performed parallel studies in human (h)LSECs). Results: PLCs and hLSECs can both be transduced (LV-lcoET3) with very high efficiency and produce high levels of biologically active FVIII. Surprisingly, both cell types were largely refractory to CRISPR/Cas9-mediated knockin of the lcoET3 fVIII transgene in the AAVS1 genome locus. However, successful insertion of an RFP reporter into this locus using an identical procedure suggests the failure to achieve knockin of the lcoET3 expression cassette at this site is likely a function of its large size. Importantly, using plasmids, alone or to introduce the CRISPR/Cas9 "machinery", resulted in dramatic upregulation of TLR 3, TLR 7, and BiP in PLCs, compromising their unique immune-inertness. Discussion: Although we did not achieve our primary objective, our results validate the utility of both PLCs and hLSECs as cell-based delivery vehicles for a fVIII transgene, and they highlight the hurdles that remain to be overcome before primary human cells can be gene-edited with sufficient efficiency for use in cell-based gene therapy to treat HA.

Comparative cultivation of single cell derived placental mesenchymal stromal cells in vitro / 上海交通大学学报（医学版）

Objective • To explore optimal placental derived mesenchymal stem cells (PMSCs) culture medium using different combination of human platelet lysate (HPL), basic fibroblast growth factor (bFGF) and traditional fetal bovine serum (FBS) for further basic and clinical study. Methods • Single cell derived PMSCs was harvested and incubated with 4 kinds of culture, i.e, FBS, FBS+bFGF, HPL and HPL+bFGF. The morphology, growth state, cell phenotype and multi-energy differentiation were observed. Cells of P1, P2, P3 and P4 generation were counted respectively. The number of units of colony formation was analyzed by inoculating P4 cells. Results • It was identified that PMSCs had the biological properties of MSCs. Quantificationally, cell density reached (1.12×107) cells/cm2 in FBS+bFGF group and (1.24×107) cells /cm2 in HPL group (P>0.05) in P4, while those in FBS group and HPL+bFGF group were (5.58×106) cells /cm2 and (8.56×106) cells /cm2, respectively. For cell morphology, the P4 PMSCs of FBS+bFGF and HPL groups kept adherent growth, but the HPL cells could not be adherent in P5 generation. The number of colony was 51/well in FBS+bFGF group, and it was 52/well in HPL group (P>0.05) in P4. The FBS+bFGF group and the HPL group were able to maintain the characteristics of MSCs and the ability of pluripotent differentiation in the P4 generation. Conclusion • PMSCs in P4 cultured in HPL medium can keep the biological characteristics and meet the clinical transplantation requirements in quality and quantity, which are preferable for their low immunogenicity to clinical applications. In long term, PMSCs cultured in FBS+bFGF medium are preferable for the longer lasting characters of MSCs and larger quantity in basic studies. HPL+bFGF medium has no advantage on quality and quantity.