Laminin as a Potent Substrate for Large-Scale Expansion of Human Induced Pluripotent Stem Cells in a Closed Cell Expansion System

Abstract

The number of high-quality cells required for engineering an adult human-sized bioartificial organ is greater than one billion. Until the emergence of induced pluripotent stem cells (iPSCs), autologous cell sources of this magnitude and with the required complexity were not available. Growing this number of cells in a traditional 2D cell culture system requires extensive time, resources, and effort and does not always meet clinical requirements. The use of a closed cell culture system is an efficient and clinically applicable method that can be used to expand cells under controlled conditions. We aimed to use the Quantum Cell Expansion System (QES) as an iPSC monolayer-based expansion system. Human iPSCs were expanded (up to 14-fold) using the QES on two different coatings (laminin 521 (LN521) and vitronectin (VN)), and a karyotype analysis was performed. The cells were characterized for spontaneous differentiation and pluripotency by RT-PCR and flow cytometry. Our results demonstrated that the QES provides the necessary environment for exponential iPSC growth, reaching 689.75 × 10⁶ ± 86.88 × 10⁶ in less than 7 days using the LN521 coating with a population doubling level of 3.80 ± 0.19. The same result was not observed when VN was used as a coating. The cells maintained normal karyotype (46-XX), expressed pluripotency markers (OCT4, NANOG, LIN28, SOX2, REX1, DPPA4, NODAL, TDGFb, TERT3, and GDF), and expressed high levels of OCT4, SOX2, NANOG, SSEA4, TRA1-60, and TRA1-81. Spontaneous differentiation into ectoderm (NESTIN, TUBB3, and NEFH), mesoderm (MSX1, BMP4, and T), and endoderm (GATA6, AFP, and SOX17) lineages was detected by RT-PCR with both coating systems. We conclude that the QES maintains the stemness of iPSCs and is a promising platform to provide the number of cells necessary to recellularize small human-sized organ scaffolds for clinical purposes.

Figure 1

Experimental design of hiPSC expansion. The QES hollow fibers were coated with LN521 or VN for at least 4 hours. Human iPSCs were loaded in the QES on day 0 in a density of >35 × 10⁶ cells in 100 ml of TeSR™ E8™ media with 10 μM Y-27632. Cells were fed via the media inlet line from days 1–6/7, at which time the cells were harvested, frozen, and/or characterized for pluripotency and differentiation markers.

Figure 2

Human iPSC after expansion in the QES. (a) Comparison between predicted and counted cells harvested from the QES. (b, c) Bright field of iPSC cultivated in LN521 and VN 3 (b) and 7 (c) days after harvesting from the QES. (d) Lactate-predicted cell number during expansion in QES. (e) Media consumption during expansion in QES. (f) Population doubling level (PDL) of hiPSCs during QES expansion; red: LN521 coating (n = 4); black: vitronectin coating (n = 3). (g) Karyotyping of hiPSCs after expansion, ^∗∗∗ p < 0.0001.

Figure 3

Pluripotency characterization after QES expansion: (a) RT-PCR for pluripotency transcripts of cells cultivated on Matrigel (CTRL1), LN521 (CTRL2), and VN (CTRL3) both in 2D culture and after the QES expansion (quantum 1—LN521 and quantum 2—VN). GAPDH was used as endogenous control. (b) Representative dot plots of pluripotency markers after QES expansion: OCT4 (CTRL 1: 94.3%, quantum 1: 97.2%, quantum 2: 97.9%), NANOG (CTRL 1: 75.8%, quantum 1: 93.3%, quantum 2: 99.1%), SOX2 (CTRL 1: 99.9.%, quantum 1: 97.6%, quantum 2: 99.6%), SSEA4 (CTRL 1: 100%, quantum 1: 100%, quantum 2: 100%), TRA1-60 (CTRL 1: 97.1%, quantum 1: 97.6%, quantum 2: 98.6%), and TRA1-81 (CTRL 1: 99.3%, quantum 1: 97.5%, quantum 2: 99.7%). Blue represents isotype controls.

Figure 4

Spontaneous differentiation of iPSCs after expansion in QES. (a) Floating embryoid bodies (EB) 7 days after aggregation. (b) Colony morphology after attachment. (c) Expression of markers of three embryonic germ layers by RT-PCR when cultivated on Matrigel (CTRL1) or after QES expansion on LN521 and VN. Scale bars represent 100 μm.