cGMP-Manufactured Human Induced Pluripotent Stem Cells Are Available for Pre-clinical and Clinical Applications

Abstract

The discovery of induced pluripotent stem cells (iPSCs) and the concurrent development of protocols for their cell-type-specific differentiation have revolutionized our approach to cell therapy. It has now become critical to address the challenges related to the generation of iPSCs under current good manufacturing practice (cGMP) compliant conditions, including tissue sourcing, manufacturing, testing, and storage. Furthermore, regarding the technical challenges, it is very important to keep the costs of manufacturing and testing reasonable and solve logistic hurdles that permit the global distribution of these products. Here we describe our efforts to develop a process for the manufacturing of iPSC master cell banks (MCBs) under cGMPs and announce the availability of such banks.

Figure 1

An Overview of the Manufacturing of Human Induced Pluripotent Stem Cells under cGMPs A tissue acquisition program was established, focusing on defining tissue requirements, working with a tissue recovery agency, establishing forms and standard operating procedures, recovering tissue, and donor eligibility determination. The manufacturing started with the isolation of CD34+ cells from a fresh cord blood unit and continued to priming, expansion, and then reprogramming of CD34+ cells. After generation of iPSCs and expansion, the cells were banked and eventually tested. Every step of the manufacturing process was documented and performed according to the batch records and standard procedures. Following characterization of the final bank, the results were reviewed by the quality assurance group to release the GMP iPSC lot.

Figure 2

Key Steps of the Human iPSC Manufacturing Process The manufacturing of human iPSCs under defined and cGMP conditions include tissue acquisition to obtain a fresh cord blood unit, CD34+ cells isolation from the cord blood, reprogramming of CD34+ cells into iPSCs using the 4D nucleofector system and episomal-based technology, and expansion and banking of iPSCs. The photomicrographs represent CD34+ cells isolated from cord blood and expanded in culture (CD34+ Isolation and Priming), iPSC colonies on day 10 post-nucleofection (P0 Colonies), and iPSCs at passage 6 (P6 Colonies) passage 12 (P12 Culture). Scale bars, 500 μm, except in the CD34+ Isolation and Priming image (250 μm).

Figure 3

Generation, Expansion, and Characterization of Human iPSCs: Engineering Runs—LiPSC-ER2.2 (A) Priming of CD34+ cells isolated from cord blood unit and expanded in culture on day 4 prior to nucleofection (Priming), the iPSC colony emerged on day 4 post nucleofection (D4 Post-Nucleofection), iPSC colonies on day 11 post nucleofection (P0 colonies), iPSCs at passage 3 (P3 colonies), and iPSCs at passage 14 (P14 culture). Scale bars, 500 μm, except in the Priming image (250 μm). (B) iPSCs stained positively with OCT4, TRA-1-60, SSEA4, NANOG, TRA-1-81, and AP. Scale bars, 250 μm, except in the AP image (500 μm). (C) iPSCs expressing the pluripotent stem cell surface markers SSEA4, TRA-1-60, and TRA-1-81 (dark blue). Light blue indicates the isotype control. (D) iPSCs differentiated into embryoid bodies and readily expressing the markers for early ectoderm (TUJ1), endoderm (AFP), and mesoderm (SMA). Scale bars, 125 μm. (E) The iPSCs demonstrated a normal karyotype after 14 passages. (F) STR analysis showed that the iPSCs matched the starting CD34+ donor sample.

Figure 4

cGMP Manufacturing of Human iPSCs: GMP Runs—LiPSC-GR1.1 (A) CD34+ cells isolated from afresh cord blood unit expanded and further purified from days 0–4 in the priming step. Black represents the CD34+ cell population, and gray represents the isotype control. (B) An iPSC colony on day 10 post-nucleofection (D10 P0 Colonies), iPSCs at passage 3 (P3 Colonies), and iPSCs at passage 13 (P13 Culture). Scale bars, 500 μm. (C) iPSCs stained positively with OCT4, TRA-1-60, SSEA4, NANOG, TRA-1-81, and AP. Scale bars, 250 μm, except in the AP image (500 μm). (D) iPSCs expressing the pluripotent stem cell surface markers SSEA4, TRA-1-60, and TRA-1-81 (dark blue). Light blue indicates the isotype control. (E) A dendrogram developed through whole gene expression analysis, confirming the clustering of the iPSC lines generated in this work and lines published previously. The colored lines indicate iPSC clones generated from the same donor. F and M indicate iPSC generation from female and male donors, respectively. (F) The iPSCs demonstrated a normal karyotype after 14 passages in culture. (G) STR analysis showed that the iPSCs matched the starting CD34+ donor sample.

Figure 5

Validation of Neural Differentiation and Gene Targeting and Preparation of the Human iPSC MCB and WCBs (A and B) Use of iPSCs (LiPSC-TR1.2) generated using a cGMP-compatible process in pre-clinical studies for neural differentiation (A) and genetic engineering (B). (A, a–c) Neural rosettes formed via EBs were isolated manually and expanded to a homogenous NSC population. Immunocytochemical analysis showed positive expression of the NSC markers SOX1 and NESTIN. Scale bars, 200 μm, except in b and c (100 μm). (B, a–c) TALEN-mediated homologous recombination targeting the safe harbor site AAVS1 on chromosome 19. A representative example of a GFP-positive clone is shown. GFP was driven by the constitutively active CAG promoter. Scale bars, 200 μm. (C) Human iPSCs generated under cGMP conditions can be used as MCB seed stocks to create working cell banks under both the GMP setting (for manufacturing specialized cell therapy products) and in a non-GMP environment (to carry out research studies for multiple cell therapy applications).