Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications

Abstract

We have recently described manufacturing of human induced pluripotent stem cells (iPSC) master cell banks (MCB) generated by a clinically compliant process using cord blood as a starting material (Baghbaderani et al. in Stem Cell Reports, 5(4), 647-659, 2015). In this manuscript, we describe the detailed characterization of the two iPSC clones generated using this process, including whole genome sequencing (WGS), microarray, and comparative genomic hybridization (aCGH) single nucleotide polymorphism (SNP) analysis. We compare their profiles with a proposed calibration material and with a reporter subclone and lines made by a similar process from different donors. We believe that iPSCs are likely to be used to make multiple clinical products. We further believe that the lines used as input material will be used at different sites and, given their immortal status, will be used for many years or even decades. Therefore, it will be important to develop assays to monitor the state of the cells and their drift in culture. We suggest that a detailed characterization of the initial status of the cells, a comparison with some calibration material and the development of reporter sublcones will help determine which set of tests will be most useful in monitoring the cells and establishing criteria for discarding a line.

Keywords: Consent; Embryonic stem cells; Induced pluripotent stem cells; Manufacturing; Markers; cGMP.

Fig. 1

Generation, expansion, and characterization of human iPSCs (LiPSC-ER2.1) - engineering runs. Panel a illustrates priming of CD34+ Cells isolated from cord blood unit and expanded in culture on day 3 prior to the nucleofection (Priming), iPSC colony emerged on day 9 post nucleofection (D9 Post-Nucleofection), iPSCs at passage 6 (P6 Colonies), and iPSCs at passage 18 (P18 culture). Panel b illustrates iPSCs positively stained with OCT4, TRA-1-60, SSEA4, NANOG, TRA-1-81, and alkaline phosphatase (AP). Panel c shows the iPSCs expressing the pluripotent stem cell surface markers SSEA4, TRA-1-60, and TRA-1-81 (dark blue). Light blue exhibits the isotype control. Panel d shows iPSCs differentiated into embryoid bodies and readily expressed the markers for early ectoderm (TUJ1), endoderm (Alpha-Feto Protein (AFP)), and mesoderm (Smooth Muscle Actin (SMA)). DAPI shows the nuclei stain in blue. The iPSCs demonstrated normal karyotype after 17 passages (e). STR analysis showed that the iPSCs matched the starting CD34+ donor sample (f). Scale bar in all images in Panel a is 500 microns except the Priming image which is 250 microns. Scale bar in all images in Panel b is 250 microns except the AP image which is 500 microns. Scale bar in all images in Panel D is 125 microns

Fig. 2

Human iPSC manufacturing process flow diagram with in process testing of samples. The process for manufacturing of human iPSCs under defined and cGMP conditions include (1) isolation of CD34+ cells from fresh cord blood unit, (2) priming CD34+ cells for 4 days, (3) reprogramming of CD34+ cells into iPSCs using 4D Nucleofector system and an episomal based technology, (4) isolation of about 9 iPSC colonies, serial subculturing of iPSCs up to 6 passages, (5) in process cryopreservation of all iPSC colonies to select the two best iPSC colonies based on the results of in process control IP-QC3, (6) expansion of two selected iPSC colonies and confirmation of plasmid clearance, (7) further expansion into large tissue culture flasks, (8) banking, and (9) final characterizations and QC testing. Multiple in-process samples (shown in green) were taken at different stages of the process and submitted for relevant testing. Each in-process test has been described in the figure. The number of in-process samples exhibits the number of tests carried out at each step

Fig. 3

Pluritest analysis of test and control samples. a. Model-based multi-class pluripotency score: pluripotency score between red lines indicates a 95 % pluripotency signature. Samples between blue line indicates 95 % of non-pluripotent samples. Line PR1.0, TR1.1, TR1.2, GR1.1, GR1.2, ER2.1, ER2.2, ER2.3, XCL1iPSC and H14 ESC are all localized between or close to the red lines, hence all pluripotent. The CD34⁺ cord blood line and ER2.2 NSC are (negative controls) are located between the blue lines demonstrates non-pluripotent signature. b. Novelty Score: This score is based on well-characterized pluripotent samples, color-coded green and non-pluripotent samples color-coded red. All iPSC and ES samples are in green demonstrates pluripotent samples, whereas CD34⁺ cord blood line and ER2.2 NSC demonstrates non-pluripotent color-code red. c. Hierarchical Clustering of vst-transformed samples: Samples were transformed using variance stabilizing transformation (VST) and robust spline normalization _ENREF_35[40]. Distance on x -axis is based on Pearson correlations. d. Overview: This combines novelty score on X-axis and Pluripotency score on y-axis. The red background, where the iPSC and ESC samples are located, suggest the empirical distribution of pluripotent cells. CD34⁺ cord blood line and ER2.2 NSC are located closer to the non-pluripotent background colored blue

Fig. 4

Whole Genome analysis conducted on two iPSC lines generated using the cGMP compliant manufacturing process. a. WGS data characterisation pipeline. This figure outlines the work flow followed in this study. The filters applied at various stages have been mentioned in the methods section. The fastq files were aligned using Isaac aligner to generate bam file. The bam file generated was checked for its mapping quality using samstat. The fastq files were also used for prediction of HLA types using HLAVBseq. The variants called by Issac variant caller was annotated using SnpEff and this data was used for predicting blood groups using BOOGIE. Only non-synonymous variants were considered for its implication with PD and cross validated with expression data. Structural variation (deletions and duplications) were filtered (see methods) and annotated for genes using UCSC. This data was cross validated with expression and microarray data. The differentially expressed genes were verified for gene enrichment relevant to disease and pathway through DAVID. b. The x-axis shows the various chromosomes and Y-axis represents the max-depth computed by Issac Variant Caller across each chromosome on a log scale for both the cell lines under study. c. Bar graph representing number of variants identified for SNPs, small Insertions, small deletions, synonymous, non-synonymous variants, CNVs and different types of structural variants including duplications, large insertions (length > 50), large deletions (length > −50), inversions and translocations. Deletions were higher in number than other types of SVs

Fig. 5

Extracting blood type and HLA from the whole genome sequencing data. a. Table showing the blood group predicted by BOOGIE for four different blood group systems namely ABO, Rh, MN and Rh associated glycoprotein with the score for both the cell lines. The validity of this approach is provided as means of comparison with the actual available donor information. b. Table showing HLA types predicted in-silico by HLAVBseq using WGS data for both the cell lines along with their depth. The predicted HLA type obtained using the WGS data is compared with the results obtained using the HLA AssureTM SE SBT kit