Fig. 9

1 The comparison between the growth of undifferentiated human pluripotent stem cells on chemically optimized substrates and feeder-containing substrates. The figure includes multiple panels showing different aspects of the experiment. Panel A depicts a schematic diagram of the UV treatment process, while panels B and C display XPS spectra indicating the surface chemical functionality of a polystyrene culture dish before and after UV treatment. Phase-contrast and fluorescence images of transgenic Oct4-GFP hESCs on these surfaces are shown, with bright green fluorescence indicating strong expression of the pluripotency marker Oct4. Panel D presents the relative number of hESC colonies on UVPS (UV-treated polystyrene) compared to conventional TCPS (standard tissue culture polystyrene) on the seventh day after cell seeding. Panel E demonstrates colony formation on virgin polystyrene treated with various UV doses, and the inset shows the prediction of colony numbers based on a PLS model. In panel F, the PLS model is used to identify surface ions that either support or inhibit hESC colony formation based on ToF–SIMS data. Panel G presents the number of adhered cells after 24 h of culture on UVPS coated with either human serum or recombinant human vitronectin, along with integrin-blocking antibodies. The results show that blocking αvβ5 integrin reduces adhesion, while blocking β1 integrin has minimal effect. Finally, panel H compares the number of undifferentiated Oct4-GFP-positive hESCs per well on UVPS and standard mouse embryonic feeder (mEF)-containing substrates after seven days of culture. UVPS coated with vitronectin is represented by the red bar, while the gray bar represents the mEF-containing substrates. The error bars in all panels indicate 95% confidence intervals, and the experiments were conducted with a sample size of three. 2 The optimization of polystyrene substrates through chemical and geometrical modifications. Panel A provides a schematic representation of the UV treatment process, which can be controlled spatially by inserting a photomask between the UV source and the dish. An overlay of phase-contrast and fluorescent images shows transgenic Oct4-GFP hESC cultures on a UV-patterned polystyrene substrate. The substrate, referred to as UV-Pattern, was coated with FBS (fetal bovine serum). Panel B presents a Time-of-Flight Secondary Ion Mass Spectrometry (ToF–SIMS) scan of the UVPS (UV-patterned polystyrene) surface after patterning with a photomask. The scan reveals the intensity of all positive ions, with different colors indicating varying intensities. The profile demonstrates a resolution of 30 μm between the points where the ion intensity changes from 20 to 80%. The abbreviation “Max.” stands for maximum. Panel C shows immunostaining of pluripotency markers in cells cultured on the UV-Pattern described in Panel A. Panel D demonstrates the possibility of patterning human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) using different geometries, suggesting versatility in the patterning process. Panel E presents the results of the experiment, showing the number of undifferentiated Oct4-GFP-positive cells in each well after 7 days of culture. The measurement was performed using flow cytometry on constant area patterns. Each well initially contained 15,000 cells, and the cumulative UV-treated area per well remained the same across all patterns. Error bars represent the 95% confidence intervals, and the surfaces were precoated with 20% bovine serum. The cells were seeded in the presence of a ROCK inhibitor for the first 8–12 h. 3 The results of simulating cell behavior on substrates patterned with UV light. In the first part (A), snapshots of human embryonic stem cells (hESCs) or hiPSCs are shown on spots with diameters of 300 and 1,400 μm. These snapshots were taken during the simulation and demonstrate that the majority of cells aggregate within 3 h, which is consistent with observations made during live imaging. The second part (B) presents the distribution of cells within each aggregate as predicted by the cell migration model. This prediction is shown for two different patterned spot diameters: 300 μm and 1,400 μm. It is important to note that no ROCK inhibitor was present in the media during these simulations. Lastly, part (C) provides information about the percentage of cells that exist as single cells, not paired or in colonies, as a function of cell density. When cells are seeded at a typical density used in routine cell culture (60,000 cells per well in a 6-well plate), the data indicates that less than 0.01% of cells remain as single cells. 4 The use of a UV-patterned substrate to facilitate long-term culture of cells. Panel A shows an overlay of phase-contrast and fluorescent images of transgenic Oct4-GFP BG01 hESC cultures on the UV-patterned substrate after 10 passages using collagenase dissociation. Panel B presents flow cytometry data of cells after two consecutive passages on the UV-patterned substrate, indicating the relative fluorescent units (RFU) and maximum (Max) values. The passage number (p) is also mentioned. Panel C displays immunostaining results for pluripotency markers in cells grown on the UV-patterned substrate. Panel D exhibits the formation of teratomas in immunodeficient mice by cells cultured on the UV-patterned substrate. Hematoxylin and eosin (H&E) staining of the teratoma reveals the presence of tissues representing all three germ layers. Panel E depicts the number of undifferentiated Oct4-GFP-positive hESCs over three passages using accutase on the UV-patterned substrate (red) compared to standard mouse embryonic feeder (mEF)-containing substrates (gray) when seeded with 24,000 cells per well of a six-well plate. The error bars indicate 95% confidence intervals, and the high R2 coefficient of determination suggests a good fit to the exponential growth model. Panel F shows flow cytometry data for pluripotency markers SSEA-4 and Tra-1–60 after more than 10 consecutive passages on the UV-patterned substrate for two different hiPSC lines, P237.1 and P237.5. Collagenase passaging (Col) is mentioned. In the case of transgenic Oct4-GFP BG01 hESCs passaged on MEFs, only GFP-positive cells were analyzed for Tra-1–60 and SSEA-4 expression, excluding MEFs from the analysis. 5 The utilization of UV-patterned substrates to facilitate the reprogramming and gene modification of human pluripotent stem cells. In panel A, phase-contrast images display BG01 human embryonic stem cells (hESCs) on UV-patterned substrates with specific dimensions. These cells were subjected to electroporation with CAAGS-GFP targeting and ZFN plasmids. Following electroporation, the cells were initially cultured in the presence of ROCK inhibitor. A successful targeted clone was then transferred to mouse embryonic fibroblasts (mEFs) and exhibited a high level of green fluorescent protein (GFP) expression after more than two months of culture. Panel B includes phase-contrast and immunostained images of “patient-237” fibroblasts on UV-patterned polystyrene. These fibroblasts were infected with a modified version of the pHAGE-STEMCCA vector, which contains loxP sites for Cre-mediated excision. The patterned surface was coated with human serum, enabling fibroblasts to adhere to the untreated areas of the dish. Over a period of four weeks, the fibroblasts underwent morphological changes and formed colonies of hiPSCs on the UV-patterned substrates. Panel C depicts the immunostaining of pluripotency markers in the patient-237 hiPSC line grown on the UV-patterned substrate. In panel D, Southern blot analysis of genomic DNA from various patient-237 hiPSC lines is shown, focusing on the Klf4 gene. The analysis reveals different bands representing the presence or absence of the reprogramming vector. The red-labeled cell lines indicate successful excision of the reprogramming vector upon Cre-recombinase expression. The loss of specific viral KLF4 bands indicates the isolation of vector-free hiPSCs through clonal selection. The accompanying bar graph illustrates the percentage of cells positive for pluripotency markers SSEA-4, TRA-1–60, and TRA-1–81, as determined by flow cytometry in a vector-free patient-237 hiPSC line after two passages on the UV-patterned substrate. Error bars represent the 95% confidence intervals based on three replicates. “Pos” denotes positive. Panel E showcases teratoma formation in immunodeficient mice resulting from the injection of vector-free hiPSCs that were reprogrammed and cultured on the UV-patterned substrate. Hematoxylin and eosin (H&E) staining of the teratoma reveals the presence of tissues representative of all three germ layers. 6 The use of a UV-patterned substrate to facilitate the transfer of individual human pluripotent stem cells. In panel A, images show BG01 human embryonic stem cells (hESCs) cultured on the “UV-Pattern” substrate after 7 and 27 passages using single-cell accutase dissociation. The image at passage 7 contains a fluorescent overlay indicating high expression of the Oct4-GFP marker. Panel B displays patient-237 hiPSCs at passage 27, with immunostaining indicating expression of the pluripotency marker Nanog (green) in all cell nuclei and high expression of SSEA-4 (red). The surfaces of the substrates were coated with 20% bovine serum, and cells were seeded in the presence of a ROCK inhibitor for the initial 8–12 h. Panel C presents flow cytometry results of BG01 hESCs with the Oct4-GFP reporter after three consecutive passages on the UV-Pattern substrate using accutase. Panel D shows flow cytometry results for pluripotency markers SSEA-4 and Tra-1–60 in cells from five different cell lines after more than 10 consecutive passages on the UV-Pattern substrate. The letter “A” denotes accutase-mediated passaging. In the case of transgenic Oct4-GFP BG01 cells passaged on mouse embryonic fibroblasts (mEFs), only GFP-positive cells were analyzed for Tra-1–60 and SSEA-4 expression, excluding mEFs from the analysis. Panel E demonstrates that patient-237 hiPSCs propagated on the UV-Pattern substrate for over 5 months (27 passages) maintained a normal 46XY karyotype. Lastly, panel F provides the design parameters used to develop the UV-treated culture system for human pluripotent stem cells. Reprinted from [527] with permission from the PNAS