Human-Induced Pluripotent Stem Cells Manufactured Using a Current Good Manufacturing Practice-Compliant Process Differentiate Into Clinically Relevant Cells From Three Germ Layers

Abstract

The discovery of reprogramming and generation of human-induced pluripotent stem cells (iPSCs) has revolutionized the field of regenerative medicine and opened new opportunities in cell replacement therapies. While generation of iPSCs represents a significant breakthrough, the clinical relevance of iPSCs for cell-based therapies requires generation of high-quality specialized cells through robust and reproducible directed differentiation protocols. We have recently reported manufacturing of human iPSC master cell banks (MCB) under current good manufacturing practices (cGMPs). Here, we describe the clinical potential of human iPSCs generated using this cGMP-compliant process by differentiating them into the cells from all three embryonic germ layers including ectoderm, endoderm, and mesoderm. Most importantly, we have shown that our iPSC manufacturing process and cell culture system is not biased toward a specific lineage. Following controlled induction into a specific differentiation lineage, specialized cells with morphological and cellular characteristics of neural stem cells, definitive endoderm, and cardiomyocytes were developed. We believe that these cGMP-compliant iPSCs have the potential to make various clinically relevant products suitable for cell therapy applications.

Keywords: cell therapy; current good manufacturing practices; differentiation; induced pluripotent stem cells; regenerative medicine.

Figure 1

Characterization of current good manufacturing practice-compliant human-induced pluripotent stem cell (hiPSC) lines (LiPSC 18R and LiPSC ER2.2). (A) LiPSC 18R positively stained with pluripotency specific markers Oct4, Nanog, Tra-1-81, SSEA4, and Tra-1-60. hiPSCs were also positive for alkaline phosphatase (AP) (top left). (B) LiPSC 18R were induced to spontaneously differentiate into three germ layers through embryoid bodies (EB) formation. Differentiated cells expressed the markers for endoderm [Alpha-Feto Protein (AFP)], mesoderm [Smooth Muscle Actin (SMA)], and early ectoderm (beta-III-Tubulin or TUJ1). (C) Flow-cytometry analysis showed that the LiPSC 18R expressed the pluripotent stem cell surface markers including TRA-1-60, SSEA-4, and TRA-1-81 (dark blue). Light blue exhibits the isotype control. (D) LiPSC 18R demonstrated normal karyotype after eight passages in culture as shown by G-banding analysis. (E) LiPSC ER2.2 positively stained with pluripotency specific markers Oct4, Nanog, Tra-1-81, SSEA4, and Tra-1-60. hiPSCs were also positive for alkaline phosphatase (AP) (top left). (F) LiPSC ER2.2 were induced to spontaneously differentiate into three germ layers through EB formation. Differentiated cells expressed the markers for endoderm (AFP), mesoderm (SMA), and early ectoderm (beta-III-Tubulin or TUJ1). (G) Flow-cytometry analysis showed that the LiPSC ER2.2 expressed the pluripotent stem cell surface markers including TRA-1-60, SSEA-4, and TRA-1-81 (dark blue). (H) LiPSC 18R demonstrated normal karyotype after 15 passages in culture as shown by G-banding analysis.

Figure 2

Both current good manufacturing practice-compliant LiPSCs lines (18R and ER2.2) differentiated into beating cardiomyocytes in two-dimensional (2D) culture system. (A) starting with good quality cells (around 100% confluency with very low differentiated areas), both hiPSC lines started morphological changes from day 1 onward until day 14. The first beating observed for both lines around days 6–7 within several patches. A large beating area has been shown for each line on days 10 and 14 (red circles). (B) All of the beating areas on day 14 of differentiation were positively stained for cardiac-specific troponin (cTnT) marker for both hiPSC lines. A representative large cardiac patch (a few millimeter size) has been shown here for each line. (C) LiPSC 18R was characterized for more cardiomyocyte markers (Desmin, myosin light chain 2, and Actin) and stained positive for cardiac progenitor marker (NKX2.5). Scale bar: 100 µm.

Figure 3

Optimization of cardiomyocyte differentiation through modulation of CHIR99021 concentration. (A) Morphology of LiPSC 18R-derived differentiated cells was changed enormously during the first week of directed differentiation. The cells with 8 µM of CHIR resulted in lots of cell death and empty spaces with no beating area observed. Dark brownish areas are representative of cardiac beating patches. (B) Immunofluorescent staining of differentiated cells for cardiomyocyte specific marker cardiac-specific troponin (cTnT) demonstrated that 4 µM of CHIR99021 resulted in higher number and bigger beating areas among the four conditions tested. (C) Flow cytometry for cTnT also confirmed the higher number of cardiomyocytes in the 4-µM condition (pink: target, blue: isotype). Scale bar: 100 µm.

Figure 4

Differentiation of LiPSCs into neural stem cells (NSCs). (A) Morphology of LiPSC 18R-derived cells was changing during the differentiation from P0 to P3. Red circle shows the neural rosette-like structure at the end of P0. (B) Immunofluorescent staining of NSCs at the end of P3 revealed that they express NSC-specific markers (Nestin and Pax6). (C) Flow-cytometry analysis at the end of P3 demonstrated that around 83.5% of cells acquired NSC morphology. Scale bar: 100 µm.

Figure 5

Differentiation of LiPSCs into definitive endoderm (DE). (A) Morphology of LiPSC-derived cells was changing during the differentiation from day 1 to day 5. The cells were highly proliferative during the differentiated period and produced dense single-layered sheets. (B) Flow-cytometry analysis at the end of day 5 demonstrated that more than 80% of LiPSC 18R-derived cells express FoxA2 and Sox17. (C) Immunofluorescent staining of day 5 cells on both lines revealed that they express DE-specific markers (FoxA2 and Sox17). Scale bar: 100 µm.