Polymerized Laminin-521: A Feasible Substrate for Expanding Induced Pluripotent Stem Cells at a Low Protein Concentration

Abstract

Laminins (LNs) play a central role in the self-assembly and maintenance of basement membranes and are involved in critical interactions between cells and other extracellular matrix (ECM) proteins. Among the defined, xeno-free ECM culture matrices, LNs-namely LN521-have emerged as promising coating systems for the large-scale expansion of induced pluripotent stem cells (iPSCs). The biologic activity of LNs is enhanced by their acidification-induced self-polymerization into a cell-associated network called polylaminin (polyLN), which can recapitulate the native-like polymeric array in a cell-free system. Here, we show for the first time to our knowledge that polyLN521 displays a native-like hexagonal-like structure and that, at basal and low concentrations, it permits the large-scale expansion of human iPSCs. Human iPSCs expanded with polyLN521 maintained the pluripotent state and showed no impairment of karyotype stability or telomere length. These results suggest that low-concentration polyLN521 is a stable and cost-effective coating for large-scale iPSC expansion.

Keywords: cell expansion; iPSC; laminin 521; pluripotency; polylaminin.

Figure 1

Structural analyses of polylaminin 521 (polyLN521). (A–C) The structure of polyLN521 is shown at progressively increasing magnifications from top to bottom rows, using laser scanning confocal fluorescence microscopy, scanning electronic microscopy (SEM,) and transmission electron microscopy (TEM). (A) A z-stack of confocal images is depicted after 3D reconstruction using the “surface” option of the ZEN software. B. Side and orthogonal views of the stacks are also shown. Panels (C–C’’) correspond to 3 optical slices selected to reveal the structure of polyLN521 at the bottom (C), middle (C’), and top (C’’) of the z-stack at higher magnification. (D,E) SEM images of polyLM521 at two magnifications. F-G. TEM images after the negative staining of polyLN521 (F) and of the plain copper grid that supports the sample (G). The polygonal-like pattern observed in (F) corresponds to the supramolecular array of the protein and not to the mesh of the grid support.

Figure 2

Expansion of human induced pluripotent stem cells (hiPSCs) under different coating conditions. (A) Representative bright-field images showing hiPSCs cultivated on basal-Matrigel, basal-LN521, and basal-polyLN521 from day 1 to day 4. (B) Representative bright-field images showing hiPSCs cultivated on low-Matrigel, low-LN521, and low-polyLN521 from day 1 to day 4. Insets represent cells with high nuclei to cytoplasm ratios. Red arrows indicate healthy colonies. Black arrow indicates dying cells.

Figure 3

Quantification of expanded human induced pluripotent stem cells (hiPSC). (A) Colony area of basal-Matrigel, basal-LN521, and basal-polyLN521 at days 2, 3 and 4 of expansion. Bar graph shows the colony area on day 4 (Matrigel, n = 41; LN521, n = 47; polyLN521, n = 44). (B) Colony area of low-Matrigel, low-LN521, and low-polyLN521 at days 2, 3 and 4. Bar graph shows the colony area on day 4 (Matrigel, n = 40; LN521, n = 62; polyLN521, n = 91). (C) Number of cells harvested on day 4 (basal- and low-Matrigel, n = 9; basal-LN521, n = 9; low-LN521, n = 7; basal and low-polyLN521, n = 9). (D) Ratio of the number of cells to the colony area (basal- and low-Matrigel, n = 9; basal-LN521, n = 9; low-LN521, n = 7; basal- and low-polyLN521, n = 9). *** p < 0.001; **** p < 0.0001.

Figure 4

Gene expression indicative of extracellular matrix–cell interactions on days 2 and 4 in human induced pluripotent stem cells (hiPSCs) cultivated on coating systems with basal and low concentrations of Matrigel, LN521, or polyLN521. (A) Expression of LAMA5. (B) Expression of ITGA3. (C) Expression of ITGA6. n = 6 at day 2, n = 5 at day 4. ** p < 0.01; *** p < 0.001.

Figure 5

Characterization of pluripotency in human induced pluripotent stem cells (hiPSCs) cultivated on polyLN521. (A,B) Genetic screening analysis: quantitative PCR (qPCR) for Chr 1q, Chr 4p, Chr 8q, Chr 10p, Chr 12p, Chr 18q, Chr 20q and Chr Xp from cells cultivated on basal-Matrigel, basal-LN521, and basal-polyLN521 ((A), n = 3) or low-Matrigel, low-LN521, and low-polyLN521 ((B), n = 3). (C) Telomere length of hiPSCs (n = 4). (D) Pluripotent gene expression in hiPSCs. (E) Representative bright-field images showing the spontaneous differentiation of iPSCs on day 2, 5 and 10 after the cells were cultivated on basal- or low-Matrigel, basal- or low-LN521 and basal- or low-polyLN521. (F) Gene expression in the endoderm, ectoderm, and mesoderm of hiPSCs cultivated on basal- or low-Matrigel, basal- or low-LN521 and basal- or low-polyLN521.

Figure 6

Large-scale expansion of human induced pluripotent stem cells (hiPSCs) using polyLN521. (A) Comparison between lactate-predicted and hemocytometer-counted cells harvested from the quantum expansion system (QES) after 6 days of expansion (data expressed in millions of cells). * p < 0.05, hemocytometer count for basal-polyLN521 vs. low-polyLN521. (B–D) Large-scale expansion characterization showing the lactate-predicted cell number (B), media consumption (C) and population-doubling level (D) of hiPSCs during expansion in QES on basal- and low-polyLN521 substrates. (E) Bright-field images of hiPSCs cultivated on basal-polyLN521 and low-polyLN521 after harvesting from the QES. * p < 0.05.