Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes

<div><p>Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed “patterned culture”), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed “non-patterned cultures”). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control.</p></div

Culture of hPSCs with SPM on rhVNT-N coated dishes.

<p>(A) Phase contrast microscopic observation of iPSC cell line PFX#9 at passage 15. (B) Expression of SSEA-3 and TRA-1-60 by flow cytometric analysis of PFX#9 in indicated culture conditions at passage 15. (C) Time course of cell proliferation in patterned dish from days 1 to 4 (left to right). (D) Cell proliferation area that was occupied inrhVTN-N-coated spot area (spot Φ = 1 mm, 0.79 mm²/spot, X axis). Plot also shows the number of spots and their areas (out of 199 rhVNT-N-coated spots, Y axis) on days 1 to 4 (left to right). (E) Microscopic observations of clump cultures, single cell non-patterned or single cell patterned cultures with the higher magnified area in red rectangles at passage 20. A representative undifferentiated clump colony is shown in the upper left photo. Scale bars are appended. (F) Time course (0–100 h) of the area occupied by PFX#9 cells (in mm²) in 5 randomly selected spots (0.79 mm²/spot) measured by captured image analysis software (ImageJ 1.450, National Institutes of Health, Bethesda, MD, USA) every hour. Average of cell occupation area at every hour is plotted as a dot. The dot graph shows representative results from 3 independent trials. (G) Cell density (cells/mm²) of PFX#9 in single cell non-patterned or in single cell patterned culture was calculated by dividing harvested cell number by 962 mm² (35-mm non-patterned culture dish) or dividing harvested cell number by 156 mm² (total 199 spots of 1 mm diameter in 35-mm patterned culture dish). The results were obtained by scoring harvested cell numbers from 18 passages of indicated cultures and are shown as a bar (mean) with error bar (standard deviation). The significance of difference between 2 groups, p = 1.45 x 10⁻⁹. Representative results of 3 independent trials are shown. (H) Growth curve of PFX#9 in non-patterned culture, patterned culture or clump culture are shown in logarithmic graphs. PFX#9 cells in patterned culture or non-patterned culture were passaged every 4 days and in clump culture on feeder-free every 6 days and on feeder (SNL) every 5 days respectively.</p

Karyotyping.

<p>Karyotype of PFX#9 maintained with SPM on rhVNT-N-coated dish (single cell patterning culture) was analyzed by mFISH at passage 5 (left). PFX#9 from single cell non-patterned culture underwent G-Band analysis at passage 15 (right).</p

Differentiation potential of hPSCs.

<p>(A) Gene expression profiles of PFX#9 cells in the indicated culture condition (clump culture, single cell non-patterned culture or single cell patterned culture) before (undifferentiated state) and after induction of differentiation via embryoid body (EB) formation. Average of gene expression values for self-renewal (undifferentiated state), ectoderm-, mesoderm- or endoderm-related genes is shown in comparison with reference standards of TaqMan hPSC Scorecard Panel (Life Technology). (B) EB formation at day 14 from PFX#9 cells (top left). EB attached to culture dish and continued to differentiate (top right). Cells were then stained with antibodies against β-tubulin (ectoderm), α-SMA (mesoderm), AFP (endoderm) and DAPI. (C) Tissue section of teratoma in NOG mouse generated by inoculating PFX#9 cells maintained in cell clumps is shown after staining with HE. Three germ layers of tissue consisting of neural rosette (ectoderm), muscle/cartilage (mesoderm) and gut-like epithelium (endoderm) are observed. Scores in Tables are visualized in bar graph below.</p

Cryopreservation and rapid thaw of KhES-1 cells grown in single cell non-patterned culture.

<p>(A) 1x10⁶ KhES-1 cells in single cell suspension from single cell non-patterned culture were cryopreserved with freezing medium, STEMCELL BANKER. The cells were thawed and cultured for 3 passages as single cells before examining the gene expression profile with TaqMan hPSC Scorecard. (B) Phase contrast image of KhES-1 cells cultured with SPM as single cells on rhVNT-N-coated dishes 3 passages after thaw. (C)Expression of SSEA-3 and TRA-1-60 on KhES-1 cells before and 5 passages after thaw was evaluated by flow cytometry. (D) Growth curve of KhES-1 in single cell non-patterned flat culture after thaw (blue line) was examined in comparison with cells in single cell non-patterned culture without cryopreservation (red line). The cryopreserved KhES-1 showed the same proliferation rate as the control in the second passage after thaw. Scores in Tables are visualized in bar graph below.</p