(P-BC-9) A Strategic Model for Generating cGMP-Compliant Induced Pluripotent Stem Cells for Cell Therapy Manufacturing: From Donor to Master Cell Bank

The demand for cGMP-compliant induced pluripotent stem cells (iPSCs) as a qualified starting material for cell therapy manufacturing continues to increase due to their robust expansion potential and ability to differentiate to numerous lineages. Interestingly, the process to generate cGMP compliant iPSCs is not well defined because it is an all-encompassing, multi-step effort that begins with donated material and ends with a master cell bank of reprogrammed cells.  

Study

Design/Methods:

To reprogram cells suitable for further manufacturing, a frozen, cord blood unit from a properly consented donor with a total nucleated cell count of 1.42 x10⁹ was selected. The infectious disease markers for the maternal sample were negative and included CHAG, CMV, HBC, HBS, HCV, HIV, HTLV, RPR and WNV. Post thaw, the erythroid progenitor population was expanded, and then reprogrammed using sendai virus expressing the Yamanaka factors POU5F1, SOX2, KLF4 and L-MYC (Cytotune™ 2.0, ThermoFisher).  Three individual clones (140101_5, 140101_8 and 140101_9) were further expanded and used to generate master cell banks (n=3). Post-cryopreservation, representative vials from each master cell bank were thawed and tested for recovery/viability, expression of pluripotency markers NANOG, POU5F1 and SOX2 was analyzed using immunofluorescence and qPCR, sendai viral clearance using qPCR, and mycoplasma using end-point PCR.  

Results/Findings:

Post-plating, the cells quickly formed colonies with tight distinct borders and a high nucleus to cytoplasm ratio (Figure 1 A and 1B, Day 2 and Day 4 post thaw, respectively). Expression of NANOG (Figure 1C), POU5F1 (Figure 1D), and SOX2 (Figure 1E) was positive, and all displayed localization to the nucleus. Quantitative gene expression for NANOG, POU5F1, and SOX2 compared to the housekeeping gene HPRT1 resulted in an average delta Ct of 0. 37±0.28, 2.15±0.69, and 2.12±0.41, respectively. In comparison to the average expression of approximately 125 human iPSC lines previously generated, gene expression of the 3 clones fell within the statistical mean (p >0.05). Using qPCR for the viral genome (SEV) and the 3 reprogramming vectors (KLF, CMY and KOS), all three clones were sendai free (Figure 1F, data shown for clone 140101_9). Finally, end-point PCR was used to ensure the clones were negative for mycoplasma (Figure 1G).

Conclusions:

Overall, our process to generate cGMP-compliant iPSCs for further manufacturing included a strategic selection of donors, a reprogramming method that is suitable for further manufacturing as sendai viruses do not incorporate into the host genome, a combination of qualitative and quantitative pluripotency analyses, and safety testing. Our data demonstrates that the selected clones were reprogrammed efficiently, and generation of master cell banks using this method is appropriate as starting material for manufacturing of a differentiated cell product.