Recently, Corning Life Sciences has developed synthetic peptide-functionalized cell culture surface, referred to as Corning® Synthemax™ that support self-renewal and differentiation of h
INTRODUCTION
Stem cells
Human embryonic stem cells (hESCs), derived from the inner cell mass of blastocysts, possess the remarkable ability to differentiate into any cell type within the human body These pluripotent cells are a vital focus of regenerative medicine and stem cell research, offering potential treatments for a wide range of diseases Their unique capacity for unlimited self-renewal and differentiation makes hESCs a promising resource for developing cell-based therapies across various medical fields.
Human embryonic stem (hES) cells possess the remarkable ability to self-renew indefinitely and differentiate into specialized cell types, offering significant potential for cell replacement therapies to repair damaged or diseased tissues As a result, hES cells are a promising source for treating conditions such as Alzheimer's disease, Parkinson’s disease, and diabetes through cell-based therapies Additionally, they are valuable for drug discovery, toxicity testing, gene therapy, and fundamental research in developmental biology However, the use of hES cells in research also raises ethical considerations and regulatory challenges.
Embryonic stem cells are ethically controversial because they are derived from surplus embryos obtained during in vitro fertilization (IVF) procedures Additionally, immune rejection remains a significant concern when using human embryonic stem (hES) cells for transplantation therapies, as the recipient's immune system may attack the transplanted cells.
Table 1 Examples for human embryonic stem cell–derived cell types (5)
Ectoderm neural precursors, dopamine neurons, motor neurons, retinal cells, keratinocytes melanocytes Mesoderm fat, cartilage, skeletal muscle, bone, blood cells, cardiomyocytes
Endoderm prostate cells, hepatocytes, lung epithelium
In 1996, a groundbreaking technology so called “Induced pluripotent stem cells technology” or
iPS cell technology was first introduced by Shinya Yamanaka's team at Kyoto University, enabling the reprogramming of adult somatic cells into embryonic-like pluripotent stem cells This breakthrough involves using four key transcription factors—Oct3/4, Sox2, c-Myc, and Klf4—delivered via retroviral vectors to convert mouse fibroblast cells into cells with properties similar to embryonic stem cells, including morphology, gene expression, epigenetic state, and differentiation potential In 2007, Yamanaka and colleagues successfully applied this method to generate human iPS cells from human fibroblasts, opening new horizons for regenerative medicine and personalized therapies.
3 generation of human iPS cells reprogramming was published in Science from Thomson’s group in the United State in 2007 (9)
Induced pluripotent stem (iPS) cells offer significant advantages over human embryonic stem (hES) cells, including the elimination of ethical concerns since they do not require the isolation of pluripotent cells from embryos Additionally, iPS cells reduce the risk of immune rejection in transplantation therapies because they can be generated directly from a patient's own cells through biopsy and reprogramming, enabling the creation of patient-specific cells This capability makes iPS cells a promising tool for developing cell-based therapies to treat many currently incurable diseases via cell replacement approaches.
History of stem cell culture technology
Culturing pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells presents unique challenges due to their requirement for specialized niches that support adhesion, self-renewal, and controlled differentiation Their inherent tendency to spontaneously differentiate complicates long-term culture management, making the maintenance of undifferentiated pluripotent stem cells a critical priority for sustaining healthy human pluripotent stem cell (hPSC) lines Ensuring proper culture conditions is essential for preventing unwanted differentiation and preserving the cells' pluripotency for research and therapeutic applications.
During the early stages of human embryonic stem (hESC) cell culture, mouse embryonic fibroblasts (MEFs) are essential as a feeder layer to support continuous self-renewal in an undifferentiated state However, preparing MEFs is time-consuming and labor-intensive, posing significant challenges Moreover, using animal-derived feeder cells raises safety concerns for clinical applications due to the potential risk of animal virus transmission.
Use human cells as a feeder layer for hES/iPS cell culture was also extensively investigated (15,
Human fetal muscle (FM), fetal skin (FS), and AFT epithelial cells were utilized as feeder layers to support the maintenance of HES3 and HES4 human embryonic stem cells (hESCs), demonstrating their effectiveness in preserving essential pluripotency features The results confirm that hESCs cultured with these feeder layers retain their characteristic morphology and ES properties, highlighting their potential for stem cell research and regenerative medicine applications.
Human embryonic stem (hES) cells are characterized by the expression of specific stem cell surface markers, maintaining normal karyotypes and pluripotency over extended cultures In our study, hES cell lines were cultured on human foreskin feeder layers for over 42 passages, successfully preserving their pluripotent properties However, reliance on feeder layers introduces potential risks of cross-contamination due to their diverse origins Therefore, developing a feeder-free and serum-free culture system is essential to ensure safer and more consistent hES cell cultivation.
Matrigel from BD Biosciences is widely used as a substrate for cultivating human embryonic stem (hES) cells in an undifferentiated state, combined with serum-free media without the need for feeder cells It is a complex mixture of extracellular matrix proteins extracted from the Engelbreth-Holm-Swarm (EHS) mouse tumor, rich in laminin, collagen IV, heparan sulfate proteoglycans, entactin, nidogen, and various undefined factors, which collectively mimic the body's extracellular environment To prepare Matrigel for cell culture, it must be thawed at 4ºC and applied to a culture plate for about an hour to form a supportive film Cultured on Matrigel, hES cells can be maintained in an undifferentiated state through over 130 population doublings while preserving normal karyotype, pluripotency markers, and high telomerase activity The mTeSR medium from StemCell Technologies is specifically formulated for use with Matrigel, facilitating an optimal culture system that sustains undifferentiated hES cell growth.
5 of hES and iPS cells in serum-free and feeder layer free conditions Compared to feeder layer cells, preparation of Matrigel coated surface is a relatively easy and inexpensive process
Matrigel is an undefined mixture of ECM proteins derived from animal tumors, leading to significant lot-to-lot variability and potential pathogenic risks Its animal origin raises safety concerns, especially for clinical applications Therefore, developing xeno-free, synthetic surfaces that can reliably replicate stem cell niches is crucial for enabling safe and effective expansion and differentiation of hES/hiPS cells in chemically defined, xeno-free culture systems.
Stem cell microenvironment
The extracellular matrix (ECM) in vivo primarily consists of macromolecules such as polysaccharides, proteins like collagens, and proteoglycans ECM is actively synthesized, secreted, and degraded by animal cells, and it is distributed either on the cell surface or between cells These macromolecules, including collagen, laminin, fibronectin, vitronectin, and elastin, form a complex network that supports and connects tissue structures while regulating crucial physiological activities of cells As a vital component of animal tissues, the ECM determines the characteristics of connective tissues and plays a key role in cell migration, differentiation, proliferation, and apoptosis.
Integrins are crucial cell surface receptor proteins that facilitate cell adhesion to the extracellular matrix (ECM) and transmit signals between the ECM and the cell, playing vital roles in cellular communication and function These heterodimeric proteins consist of α and β chains, with the α chain ranging from 120 to 185 kDa and the β chain from 90 to 110 kDa.
Twenty-seven different integrin subunit combinations have been identified so far, comprising 18 α subunits and 9 β subunits Integrins are transmembrane proteins characterized by a short cytoplasmic domain and play crucial roles in cell adhesion Both α and β subunits contain divalent cation-binding domains that regulate integrin activity; magnesium ions (Mg²⁺) enhance ligand binding, while calcium ions (Ca²⁺) inhibit this process.
Integrins on the cell surface play a crucial role in mediating cell-ECM interactions by binding to various ECM macromolecules, including collagen, laminin, fibronectin, and vitronectin A key feature of this binding process is the recognition of the RGD (Arginine-Glycine-Aspartic acid) motif, which is the most common adhesion sequence found in ECM proteins This interaction facilitates cell adhesion, migration, and signaling, highlighting the importance of integrin-ECM binding in numerous biological processes.
Integrins are essential multi-functional proteins expressed by most cells, playing a crucial role in various biological processes such as cell adhesion, proliferation, and wound healing Their ability to mediate cell-ECM interactions is critical for platelet aggregation during wound repair and for the proliferation of specific cell types Disruption of integrin-ECM interactions can impair cell attachment and spreading, highlighting their importance in maintaining cellular functions and tissue integrity.
Stem cell fate, including self-renewal, differentiation, and apoptosis, is primarily regulated by the microenvironment known as the stem cell niche, which involves critical cell-ECM and cell-cell interactions Recently, stem cell niches have been recognized as dynamic microenvironments that actively influence tissue growth and repair processes within the organism For example, the injection of fibronectin, a key glycoprotein in the extracellular matrix, can modulate stem cell behavior and enhance regenerative outcomes Understanding these niche dynamics is essential for advancing regenerative medicine and developing targeted therapies.
Fibronectin, a protein produced in the body, plays a crucial role in anchoring cells in place, which can help prevent the development of chronic pain commonly associated with spinal cord injuries (SCI) A one-time injection of fibronectin has shown promise in supporting spinal cord repair and mitigating long-term pain symptoms.
Injecting 50 μg/mL into the spinal dorsal column at a rate of 1 μL/min for a total volume of 5 μL immediately after spinal cord injury (SCI) can inhibit the development of certain types of chronic pain, such as pressure-induced pain that typically affects SCI patients Bone marrow mesenchymal stem cells (MSCs) support the hematopoietic stem cell environment in the bone marrow and serve as a niche for their own maintenance The cardiovascular progenitor cell (CPC) niche is critical for the maintenance and expansion of CPCs in developing human and mouse hearts During embryonic development and stem cell differentiation, microenvironmental factors—such as gradients of proteins like sonic hedgehog (SHH)—play a key role in guiding tissue differentiation and lineage development, although the mechanisms of stem cell niches and their activation remain largely elusive.
Wnt Pathway
The Wnt/β-catenin signaling pathway is essential for regulating cellular proliferation, cell fate decisions, and organ development It is well established that Wnt signals modulate β-catenin expression, leading to increased expression of integrins, which are crucial for cell adhesion and communication This pathway plays a pivotal role in various biological processes and is a key target for research in developmental biology and regenerative medicine.
The Wnt signaling pathway is named after the Wg (wingless) gene and the Int (integration) gene, highlighting their foundational roles (34) The wingless gene was originally discovered in Drosophila and is crucial for embryonic development, influencing cell growth and patterning In contrast, the Int gene, essential for adult tissue formation, was first identified in vertebrates and is located near the mouse mammary tumor virus (MMTV) locus, emphasizing its importance in mammalian development and disease processes.
The article discusses 8 integration sites related to vital genes such as Int-1 and wingless, highlighting their homology Mutations in the Drosophila wingless gene can cause wing deformities, while in mice, MMTV replication and genomic integration can trigger the production of one or multiple Wnt genes Wnt ligands, originating from a common ancestral gene across various organisms, interact with extracellular matrix (ECM) molecules to regulate their functions on target cells, emphasizing their crucial role in developmental processes and disease mechanisms.
The canonical Wnt pathway begins when Wnt proteins bind to Frizzled receptors on the cell surface, activating Dishevelled (DSH) proteins and leading to changes in β-catenin levels in the nucleus Activation of DSH inhibits the formation of the β-catenin destruction complex, which includes axin, GSK-3, and APC, preventing β-catenin degradation Stabilized β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it interacts with TCF/LEF transcription factors to promote the expression of Wnt target genes Understanding this pathway is crucial for insights into cell signaling and gene regulation mechanisms.
1.4.3 Regulation of stem cell by canonical Wnt pathway
Wnt pathways also play important role in maintaining stem cells in undifferentiated state, regulating proliferation of intestinal stem cells, skin stem cells and haematopoietic stem cells (36,
37, 38) Activation of canonical Wnt pathway by inhibiton of GSK3β maintains pluripotency and self-renewal of embryonic stem cells (39) Overexpression of activated β-catenin and activation
The canonical Wnt pathway plays a crucial role in stem cell biology by promoting self-renewal in long-term in vitro cultures and enhancing the reconstitution of hematopoietic lineages in vivo It also determines stem cell fate, with active Wnt3a promoting the differentiation of neural stem cells into neuronal and glial cells Interestingly, stem cells can differentiate into follicular cells without β-catenin, highlighting the pathway’s complex regulatory functions Overall, the canonical Wnt pathway is essential for both the expansion and fate determination of stem cells.
Synthetic peptide surface
Concerns over contamination and immunogenic responses from animal-derived culture components like mouse feeder cells and Matrigel have driven the shift towards xeno-free, chemically defined culture systems for human embryonic stem (hES) and induced pluripotent stem (iPS) cells Developing such defined substrates is essential for safe clinical applications, as they provide necessary niches for cell attachment and proliferation without undefined animal factors Recent advancements have focused on recombinant ECM proteins that support the survival and self-renewal of pluripotent stem cells over multiple generations, marking a significant step in tissue engineering and regenerative medicine.
Several peptides identified through phage display libraries have demonstrated the ability to support human embryonic stem cell (hESC) expansion for at least three passages, highlighting their potential in stem cell culture applications Additionally, researchers have synthesized biologically active peptides derived from key components of Matrigel, a widely used extracellular matrix substitute Notably, laminin, a major component of Matrigel, has been extensively studied, with three laminin-derived active domains showing promising results in promoting hESC growth and adhesion, thereby enhancing stem cell expansion techniques.
Recent advancements in synthetic substrates have significantly improved hESC support for self-renewal and proliferation, with peptide-coated surfaces supporting up to three to five passages Corning® Synthemax™ Surface, a synthetic peptide-functionalized culture platform, utilizes acrylate-based polymers conjugated with specific peptides to maintain hESC undifferentiated state and normal morphology Peptides derived from proteins such as vitronectin, fibronectin, laminin, and bone sialoprotein are incorporated into the surface, with the VN-PAS peptide surface demonstrating particular effectiveness in supporting hESC culture and self-renewal capabilities.
The Synthemax surface effectively supports human embryonic stem (hES) cell self-renewal and induced differentiation, maintaining cell stability over at least 12 passages with media such as mTeSR1, Knock Out Serum-supplemented medium, or TeSR2 It preserves key characteristics of stem cells, including stable doubling time, high viability, normal morphology, unaltered karyotype, and sustained expression of pluripotency markers like Oct4, TRA-1-60, and SSEA4 Additionally, differentiation experiments demonstrated the formation of teratomas comprising all three germ layers—endoderm, mesoderm, and ectoderm—as well as embryoid bodies, confirming the surface’s capability to support pluripotency and differentiation potential of hES cells.
H7 cultured after 8 passages on the Synthemax In addition, Cardiomyocytes were directly differentiated from H7 hES cells on Synthemax surface by using a protocol in previous report
The suitability of the synthetic peptide surface, Synthemax, for supporting hiPSC growth and differentiation remains uncertain While hiPSCs and hESCs share similar global transcriptome patterns, a small subset of 318 genes shows differential expression, potentially reflecting a genetic memory from their ancestor cells Assessing whether Synthemax can effectively maintain and direct the differentiation of hiPSCs is therefore crucial This study focuses on evaluating hiPSC attachment, proliferation, and long-term maintenance on Synthemax, aiming to determine its efficacy for sustained cell growth and differentiation over time.
MATERIALS AND METHODS
iPS cell culture
The human iPS cell line IMR90 was obtained from the Wicell Research Institute and maintained on growth factor-reduced Matrigel-coated dishes using the chemically defined medium mTeSR1 at 37°C with 5% CO2, with daily medium exchange To preserve the undifferentiated state, spontaneously differentiated colonies were regularly identified and removed, employing two techniques: "pick-to-remove," where differentiated colonies were physically detached and aspirated, and "pick-to-keep," where undifferentiated cells were carefully replated on a new dish For growth and differentiation analysis, approximately 5×10^4 iPS cells per cm² were seeded onto Synthemax-coated six-well plates, with Matrigel-coated wells serving as controls Daily microscopic imaging monitored cell attachment and proliferation, and cell counts were performed via Trypan-blue staining every 24 hours Cell doubling time was calculated using the formula td = ln2/α, providing insights into iPS cell growth kinetics on different substrates.
Immunofluorescence staining
Immunofluorescence staining was conducted using specific primary antibodies and fluorescent dye-conjugated secondary antibodies to accurately detect protein expression in cells Briefly, cells were thoroughly rinsed twice with 0.5 ml of ice-cold Dulbecco's Phosphate Buffered Saline (DPBS) per well to prepare for staining.
This study involved fixing cells with 4% paraformaldehyde at room temperature for 15 minutes, followed by thorough washing with ice-cold DPBS Cells were then permeabilized with 0.5% Triton X-100 in DPBS without Ca²⁺/Mg²⁺ for 10 minutes and washed three times to prepare for antibody staining Blocking nonspecific binding was achieved using a buffer containing Tween-20, Triton X-100, BSA, and DPBS for one hour Primary antibodies were applied overnight at 4°C, with secondary fluorophore-conjugated antibodies incubated in the dark at room temperature Nuclear localization was visualized with DAPI staining, and mounting was performed using VECTASHIELD with DAPI Fluorescence images were captured using an Olympus IX71 inverted microscope with a CCD camera and analyzed via Slidebook software, ensuring high-resolution visualization of cellular components.
Table 2 Primary and secondary antibodies used for immunofluorescence staining
Primary antibody company ratio Secondary antibody company ratio
Mouse monoclonal anti- human SOX17
1:100 rabbit monoclonal anti- human FOXA2
1:1000 donkey anti-rabbit IgG TRITC
1:100 rabbit anti-zyxin Sigma, St Louis, 1:100
Western blotting
Cells were cultured on Matrigel-coated and Synthemax plates for 48 hours, then harvested using Trypsin-EDTA treatment followed by centrifugation at 300×g for 5 minutes and washing with DPBS Cell pellets were lysed with a specialized buffer containing Tris, NaCl, DTT, SDS, Triton X-100, and PMSF, using a syringe and needle to ensure thorough lysis The lysates were then centrifuged at 21,000×g for 15 minutes at 4°C to separate debris, and the supernatants were collected for protein analysis Protein concentrations were quantified using the Pierce BCA protein assay kit, and cytoplasmic and nuclear proteins were extracted utilizing a dedicated extraction kit from Thermo Scientific All protein samples were properly stored at appropriate conditions for downstream applications.
2.3.2 SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)
Certain amount of cellular protein samples were mixed with 2×Laemmli loading buffer (Bio-Rad Laboratories, Inc., Hercules, CA) containing 5% of β-mercaptoethanol and heated at 98°C for 5
After heat treatment, the samples were centrifuged at 21,000 ×g for 5 minutes to ensure proper preparation The samples were then loaded onto wells of a 4-20% Mini-Protein® Precast gel (Bio-Rad Laboratories) for electrophoresis, which was conducted in Tris/Glycine/SDS running buffer at 200 V for 30 minutes The Magic Mark™ XP Western Standard (Invitrogen) served as the protein molecular weight marker to accurately determine protein sizes during analysis.
PVDF nitrocellulose membranes were prewetted in methanol for 1 minute and then soaked in transfer buffer containing 24.8 mM Tris, 192 mM Glycine, and 20% v/v methanol Following SDS-PAGE electrophoresis, the gel was carefully transferred onto a nitrocellulose membrane within a transfer cassette that included a sponge, filter paper, the gel, and additional filter paper and sponge layers Protein transfer was carried out using a Bio-Rad Tetra Cell filled with cold transfer buffer, maintained at low temperatures with an ice box The transfer was performed at 100 V for 1 hour, and subsequently, the membrane was washed twice with Tween-PBS buffer (0.05% Tween-20 in 1x PBS) to remove residual reagents.
The membrane was blocked with a buffer containing PBS, Tween-20, and non-fat milk for 1 hour at room temperature with shaking to prevent nonspecific binding Primary antibodies were then applied in blocking buffer and incubated for either 1 hour at room temperature or overnight at 4°C with shaking to ensure specific antibody binding Following three washes with Tween-PBS buffer, HRP-conjugated secondary antibodies were added and incubated for 1 hour with shaking, followed by another set of three washes Detection was achieved by incubating the membrane with Super Signal West Substrate Working Solution for 1 minute to visualize protein bands via chemiluminescence.
IL) Protein expression was detected using a Molecular Imager ChemiDoc XRS System (Bio- Rad Laboratories) and PDQuest Analysis software from Bio-Rad Laboratories, Inc
Table 3 Primary and secondary antibodies used for Western blotting analysis
Primary antibody company ratio Secondary antibody company ratio rabbit anti- vinculin
1:1000 rabbit anti-zyxin Sigma, St Louis,
Integrin blocking assay
To investigate the role of integrins in cell attachment to synthetic peptide surfaces, IMR90 cells were detached using dispase treatment followed by gentle scraping The collected cells were then washed with CMRL-BSA medium containing L-glutamine, pyruvate, and 0.35% BSA About 70,000 cells were incubated in 1 ml of CMRL-BSA medium, either in the presence or absence of anti-human integrin antibodies, to assess integrin-mediated adhesion.
Six different samples were tested, including control (without antibody), anti-α5, anti-β1, anti-α6, anti-αVβ5, and a combination of all four antibodies, with 10 µg of 1 mg/ml integrin antibodies added to each sample For the combined antibody treatment, 10 µg of each anti-integrin antibody was used Cells were seeded onto Synthemax plates and cultured for 1 hour at 37°C in a CO₂ incubator, with additional seeding on Matrigel-coated plates for comparison Following incubation, cells were washed three times with CMRL-BSA medium, fixed with ethanol, stained with crystal violet, and washed multiple times to remove excess dye Colony numbers from at least seven randomly selected regions were counted under a 10x brightfield microscope, and images were captured using an Olympus IX71 inverted phase contrast fluorescence microscope with a CCD camera and Slidebook 4.2 software All experiments were independently repeated at least three times to ensure reproducibility.
Definitive endoderm differentiation from human iPS cell
We differentiated IMR90 cells into definitive endoderm (DE) following our established protocol Cells were seeded on Synthemax plates and cultured in mTeSR1 medium until reaching 40-50% confluence, at which point they were treated with a specialized DE differentiation medium This medium comprised RPMI1640, nonessential amino acids, sodium pyruvate, B27 supplement, 1mM sodium butyrate, and 4 nM activin A After 24 hours, sodium butyrate concentration was reduced to 0.5mM, and the medium was refreshed every other day until day 7 to promote effective differentiation.
Quantitative real time–polymerase chain reaction (qRT-PCR) analysis
To assess the expression of the DE marker genes SOX17 and FOXA2 in differentiated DE tissue derived from iPS cells, total RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN) TaqMan qRT-PCR was conducted with the QuantiTecT Multiplex RT-PCR NR Kit (QIAGEN) following the manufacturer’s protocol The expression levels were normalized to the endogenous control gene cyclophilin (Applied Biosystems), a human housekeeping gene, and compared to RNA from adult human pancreata (Stratagene) To ensure the accuracy of the results, no reverse transcription control and no template control samples were included to verify the absence of genomic DNA contamination and false positives in the assay Primer-probe pairs (52) were used for specific detection of target genes.
Sox17-probe (5’FAM to 3’-Tam): ACGCCGAGGGCTACTCCTCC
Foxa2- probe (5’FAM to 3’-Tam): CAGAGCCCTCGGCACTGCC
Statistical analyses
Data were presented as mean ± standard deviation The statistical analysis was performed based on the Student’s t-test using a one-tailed algorithm The significance was determined at p 0.05
RESULTS AND DISCUSSION
Characterization of iPS cells attachment and proliferation on synthetic peptide surface 20
To assess the attachment and proliferation of iPS cells on synthetic peptide surfaces, IMR90 cells were seeded onto Synthemax plates, with Matrigel-coated plates serving as controls Results showed that cell attachment took longer on Synthemax surfaces compared to Matrigel, indicating slower adhesion After two days of culture, colonies on the synthetic peptide surface appeared rounder and were generally smaller than those on the Matrigel-coated plates These findings suggest that surface coating influences both the attachment kinetics and colony morphology of iPS cells during culture.
Figure1 IMR90 cell colony grown on Matrigel (MG) and Synthemax surface (SM)
Figure 2 illustrates the proliferation dynamics of iPS cells cultured on both Matrigel- and synthetic peptide-coated surfaces, providing insights into their growth behavior In the experiments, approximately 50,000 IMR90 cells per square centimeter were seeded onto Synthemax-modified six-well plates, with an identical cell density applied to Matrigel-coated plates for comparison The results reveal distinct proliferation patterns, highlighting the differences in cell growth kinetics between the two surface types This study underscores the impact of surface coatings on iPS cell proliferation, which is crucial for optimizing culture conditions in regenerative medicine.
21 indicated that cells grown on Synthemax are equivalent to that on Matrigel Equation 1 was used to calculate the doubling time:
Where X is the amount of cells; X 0 is the amount of cells at time 0; à is the specific growth rate; t is the culture time
The doubling times of cells cultured on Synthemax and Matrigel-coated plates were determined to be 44.05±1.45 hours and 42.98±7.86 hours, respectively, based on the slope of the linear equation derived from cell count data Our analysis shows there is no significant difference in the specific growth rates of cells on these two surfaces However, colonies on Matrigel were observed to be larger than those on Synthemax, a finding confirmed by immunofluorescence staining results presented in Figures 11 and 12.
Figure 2 (A) Growth curve of iPS cells on Synthemax and Matrigel-coated plates (B)
Estimation of the specific growth rate à Three independent experiments were conducted to calculate the slope à
In our experiment assessing cell proliferation on Synthemax surface, we observed that after more than three days of culture, iPS cells entered the exponential proliferation phase, reaching approximately 14 million cells by day 6 in a six-well plate No differentiated cell colonies were detected, indicating maintained pluripotency Unlike Matrigel-coated surfaces, where colonies grow larger and require subculturing every 3–4 days to prevent spontaneous differentiation, Synthemax supports smaller colonies and allows for longer culture periods before subculture This advantage enables more extensive expansion of iPS cells, and our results demonstrate that iPS cell productivity on Synthemax is significantly higher than on Matrigel-coated surfaces.
Figure 3 Capability of iPS cell growth on synthetic peptide surface
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Figure 4 Fluorescence microscopy images of anti-OCT4 and anti-SSEA4 labeled iPS cells
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Alexa (Alexa Fluro 488 IgG3 (1:200) were used as secondary antibodies
Figure 5 Distinct differentiated colonies grown on Synthemax plate after 13 passages under bright field Scale bar: 50 àm
Differentiation on Synthemax plate
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Figure 6 illustrates the expression of definitive endoderm (DE) marker genes and proteins in IMR90 cells differentiated on MG and SM substrates After six days of differentiation, morphological changes indicative of DE formation were observed qRT-PCR analysis revealed significant upregulation of DE markers Foxa2 and Sox17, with data shown as mean ± SD Immunofluorescence staining confirmed the presence of DE markers, utilizing mouse monoclonal anti-human SOX17 and rabbit monoclonal anti-human FOXA2 primary antibodies, followed by appropriate fluorescent secondary antibodies, highlighting the successful differentiation into definitive endoderm cells.
Functional role of integrins in iPS cell attachment
Synthemax Surface is composed of the VN-PAS surface, which facilitates cell adhesion primarily through the integrin αVβ5, as confirmed by integrin inhibition assays showing 93% reduction in iPSC attachment when αVβ5 is blocked In contrast, blocking integrins α5, α6, and β1 resulted in only minor decreases in adhesion (20%, 6%, and 11%, respectively), indicating that αVβ5 is the dominant mediator on Synthemax The combined use of all four integrin antibodies nearly completely abolished iPSC attachment, suggesting that a single integrin, αVβ5, predominantly interacts with the vitronectin-derived peptide surface These findings support the hypothesis that αVβ5 mediates recognition of the recombinant vitronectin protein on Synthemax Conversely, on Matrigel-coated plates, integrin blocking had less impact, with β1 inhibition producing the most significant reduction (~40%), highlighting the importance of β1 in cell adhesion to Matrigel and aligning with reports that integrin β1 is essential for hiPSC adhesion and proliferation on Matrigel surfaces.
The study demonstrates that a combination of antibodies targeting integrins α5, α6, β1, and αVβ5 leads to a 62% reduction in hiPSC adhesion to the Matrigel surface, indicating that multiple integrins collaboratively facilitate cell attachment These findings highlight the key roles of various integrins in mediating hiPSC adherence to extracellular matrix components like Matrigel Targeting these integrins can significantly influence hiPSC adhesion, which is crucial for stem cell culture and regenerative medicine applications.
Integrins play a crucial role in promoting iPS cell adhesion to both MG (Matrigel) and SM (Synthemax) substrates Micrographic images reveal increased cell attachment on SM surfaces when integrin antibodies are not blocked, highlighting the importance of integrin-mediated adhesion Quantitative analysis shows significantly higher iPSC attachment levels on MG and SM surfaces, with statistical significance indicated by p-values of 0.037, 0.0059, and less than 0.0001 Blocking integrins with antibodies at a dilution of 1:40 markedly reduces cell adhesion, emphasizing the essential function of integrins in iPSC attachment to these biomaterials.
Wnt pathway
The Wnt pathway is crucial for human embryonic stem (hES) and induced pluripotent stem (iPS) cell expansion and differentiation To determine whether surface substrates influence this pathway, we examined the nuclear translocation of β-catenin in iPSCs grown on SM versus MG-coated surfaces Western blot analysis revealed reduced β-catenin translocation to the nucleus in cells cultured on SM, indicating decreased activation of the β-catenin-mediated Wnt signaling pathway This suggests that the SM surface, by limiting integrin-mediated attachment and proliferation, downregulates Wnt signaling, which may transiently support iPSC proliferation, as supported by the data shown in Figures 3, 4, and 8.
Figure 8 illustrates the expression levels of cytoplasmic and nuclear β-catenin in iPSCs cultured on Matrigel (MG) and Synthemax (SM) surfaces, with protein extracts analyzed via Western blot The results, derived from two independent experiments, demonstrate the localization and abundance of β-catenin in these cells Western blotting was performed using rabbit anti-human β-catenin antibodies at a dilution of 1:2000, followed by anti-rabbit IgG HRP at 1:1000, with β-actin serving as a loading control to ensure accurate comparison.
Organization of the cytoskeleton structures
The cytoskeleton plays a crucial role in integrin-mediated signaling pathways, with integrin cytoplasmic domains connecting to the cytoskeleton via adapter proteins such as vinculin, α-actinin, and phosphorylated focal adhesion kinase (p-FAK) To understand how different substrates influence cytoskeletal organization, we analyzed the expression of actin filaments (F-actin) and vinculin during iPS cell proliferation on Synthemax compared to Matrigel-coated surfaces Using phalloidin, which specifically binds F-actin, we observed that cells on the Synthemax surface exhibited a distinct actin filament network characterized by denser and broader actin bundles between cell-cell interfaces, differing significantly from those on Matrigel Additionally, vinculin expression, pivotal in linking the cytoplasm to focal adhesions, was examined through immunostaining and Western blot analysis, revealing low vinculin levels in cells grown on Synthemax These findings suggest that substrate composition significantly impacts cytoskeletal structure and associated protein expression during stem cell culture.
Figure 9 Micrographic images of F-actin expression in cells grown on MG (A) and SM (B) surface Scale bar: 50àm Magnification: 40ì Antibody: Alexa Fluor 488 phalloidin (1:40)
Figure 10 presents micrographic images illustrating vinculin expression in cells grown on MG (A) and SM (B) surfaces, with a scale bar of 100 µm at 20× magnification Immunofluorescence staining was performed using rabbit anti-human vinculin (1:50) as the primary antibody and mouse anti-rabbit IgG-FITC (1:150) as the secondary antibody, highlighting the differences in vinculin distribution between the two surfaces These images demonstrate the cellular adhesion and cytoskeletal organization influenced by the specific surface characteristics, emphasizing vinculin's role in cell-surface interactions.
Our study demonstrates increased zyxin protein expression in cells cultured on the Synthemax surface, with Western blot analysis confirming significant upregulation Zyxin, a zinc-binding phosphoprotein located at focal adhesions and along the actin cytoskeleton, plays a crucial role in cell spreading and proliferation, and its inverse relationship with differentiation suggests that its elevation may enhance cell attachment and growth on Synthemax Additionally, α-actinin expression levels were similar in cells grown on Synthemax and Matrigel substrates, indicating comparable cytoskeletal organization The levels of phosphorylated FAK (p-FAK) showed no significant difference between cells on the two surfaces, implying that cytoskeletal reorganization and focal adhesion dynamics may facilitate iPS cell spreading and self-renewal on peptide-based substrates, despite unclear mechanisms.
Figure 11 presents micrographic images demonstrating Zyxin expression in cells cultured on MG and SM surfaces Cells grown on MG surfaces are shown in images A and C, while those on SM surfaces are displayed in images B and D, with scale bars of 100μm for A and B, and 50μm for C and D The images were captured at magnifications of 20× for A and B, and 40× for C and D Immunofluorescence staining was performed using rabbit anti-human Zyxin (1:100) as the primary antibody and mouse anti-rabbit IgG-FITC (1:150) as the secondary antibody, highlighting the cellular localization of Zyxin in response to different surface treatments.
Micrographic images reveal α-actinin expression in cells cultured on MG and SM surfaces, highlighting cellular adhesion and cytoskeletal organization Cells grown on MG surfaces (A&C) and SM surfaces (B&D) demonstrate distinct patterns of α-actinin distribution, indicative of their interactions with different substrates The images, captured at a magnification of 20× for A&B and 40× for C&D, utilize rabbit anti-human α-actinin (1:100) as the primary antibody and mouse anti-rabbit IgG–FITC (1:150) as the secondary antibody, with scale bars of 100 μm and 50 μm to denote size These findings suggest surface-dependent variations in cytoskeletal protein expression, relevant for biomaterial biocompatibility and cell adhesion studies.
Figure 13 illustrates the expression levels of key cytoskeletal proteins in iPS cells cultured on Matrigel (MG) and Synthemax (SM) surfaces Cells were harvested 48 hours after seeding, and total protein extracts were analyzed via Western blot to assess the expression of vinculin, α-actinin, and zyxin These proteins are critical markers of cytoskeletal integrity and cell adhesion, providing insights into how different surface coatings influence cytoskeletal organization in iPS cells The results highlight variations in protein expression profiles depending on the substrate, which may impact cell morphology and mechanotransduction pathways relevant to stem cell maintenance and differentiation.
N o rm a liz e d v in c u lin e x p re ss io n p=0.0002
N o rm a liz e d -a c ti n in e x p re ss io n p=0.29
Protein expression analysis was conducted using Western blotting, with β-actin serving as a loading control to ensure equal protein loading across samples Semi-quantitative analysis was performed using Kodak 1D gel imaging software, based on data from at least three independent experiments, with results expressed as mean ± SD Primary antibodies targeting vinculin, α-actinin, and zyxin were used at dilutions of 1:200, 1:1000, and 1:1000, respectively, to detect these proteins Mouse anti-rabbit IgG HRP secondary antibodies (1:1000) facilitated signal detection The bands presented are representative results from three separate experiments, confirming the reproducibility and reliability of the findings.
Micrographic images illustrate p-FAK expression in cells cultured on MG (A) and SM (B) surfaces, with a scale bar of 100 µm and magnification of 20× The study utilized rabbit anti-human p-FAK (1:50) as the primary antibody and mouse anti-rabbit IgG–FITC (1:150) as the secondary antibody to visualize FAK expression These images highlight the differences in cellular adhesion and focal adhesion kinase distribution depending on the surface type, providing insights into cell-material interactions.
CONCULSION AND FUTURE WORKS
This study evaluated the attachment, proliferation, and induced differentiation of human iPS cells on the Synthemax surface, demonstrating that iPS cell colonies grown on Synthemax exhibited a more compact morphology with less spreading compared to Matrigel Importantly, these cells maintained stable proliferation and pluripotency marker expression over ten passages, indicating the surface's suitability for long-term culture Interaction analysis revealed that iPS cells primarily utilize αVβ5 integrin to attach to the Synthemax substrate, which contains vitronectin-derived peptide sequences Additionally, reduced β-catenin activation suggests decreased Wnt signaling, contributing to the formation of more compact colonies Cytoskeleton analysis showed denser actin filament formation at cell-cell interfaces, along with down-regulation of vinculin and up-regulation of zyxin, indicating cytoskeletal remodeling Overall, these findings support that the Synthemax surface, combined with a defined medium, offers a promising, fully defined culture system for expanding clinical-grade human iPS cells for cell therapy applications.
Future experiments, including the formation of teratomas from long-term cultured iPS cells injected onto Synthemax surfaces in mice, will help further validate the efficacy of Synthemax in maintaining iPS cell pluripotency Importantly, as demonstrated in Fig 5, the Synthemax surface effectively supports the preservation of the pluripotent state of iPS cells during extended culture periods.
Our study found that spontaneous differentiation of iPS cells becomes uncontrollable after more than 12 passages on Synthemax surfaces, whereas iPS cells can be maintained in an undifferentiated state for over 40 passages on Matrigel-coated surfaces, consistent with previous research Since Matrigel contains a mixture of animal ECM proteins, multiple integrins on the iPS cell surface can bind to various ECM components, providing a robust adhesion and spreading microenvironment essential for long-term cell maintenance Future research should focus on developing chemically-defined synthetic peptide surfaces by coating multiple bioactive peptide sequences derived from ECM proteins to support the long-term expansion and directed differentiation of hES/iPS cells Additionally, detailed investigation of cytoskeletal structures and their reorganization on synthetic peptide substrates requires advanced imaging with 100x or 63x objective lenses; however, current pre-coating methods hinder the use of higher magnification The development of self-coating peptides for HPSC self-renewal and differentiation opens new avenues for understanding cell-matrix interactions and cell fate regulation influenced by synthetic peptide substrates.
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5.1 Extraction of cytoplasmic and nuclear protein
1 Harvest with trypsin-EDTA and then centrifuge at 500 × g for 5 minutes
2 Wash cells by suspending the cell pellet with PBS
3 Transfer 1-10 × 10 6 cells to a 1.5mL microcentrifuge tube and pellet by centrifugation at
4 Use a pipette to carefully remove and discard the supernatant, leaving the cell pellet as dry as possible
5 Add ice-cold CER I to the cell pellet (Table 1) Proceed to cytoplasmic and nuclear protein extraction, using the reagent volumes indicated in Table 1
6 Vortex the tube vigorously on the highest setting for 15 seconds to fully suspend the cell pellet Incubate the tube on ice for 10 minutes
7 Add ice-cold CER II to the tube
8 Vortex the tube for 5 seconds on the highest setting Incubate tube on ice for 1 minute
9 Vortex the tube for 5 seconds on the highest setting Centrifuge the tube for 5 minutes at maximum speed in a microcentrifuge (~16,000 × g)
10 Immediately transfer the supernatant (cytoplasmic extract) to a clean pre-chilled tube
11 Suspend the insoluble (pellet) fraction produced in Step 9, which contains nuclei, in ice- cold NER
12 Vortex on the highest setting for 15 seconds Place the sample on ice and continue vortexing for 15 seconds every 10 minutes, for a total of 40 minutes
13 Centrifuge the tube at maximum speed (~16,000 × g) in a microcentrifuge for 10 minutes
14 Immediately transfer the supernatant (nuclear extract) fraction to a clean pre-chilled tube
1 Cells were cultured for 48 hours on Matrigel coated plate and Synthemax plate and detached by Typsin EDTA
2 Cells were collected by centrifuged at 300 ×g for 10 min and washed by Dulbecco’s Phosphate buffered Saline
3 All cells were lysed with lysis buffer by using a 1 ml syringe with 20G1 1 /2 needle up and down 20 times
4 Cell lysates were centrifuged with 21,000 ×g at 4 o C for 15 min
5 Equal amount of cellular protein with 2×Laemmli loading buffer containing 5% of β- mercaptoethanol were heated at 98°C for 5 minutes
6 the samples were centrifuged at 21,000 ×g for 5 min Proteins were loaded into a 4-20% Mini-Protean ® Precast gel
7 Start the electrophoresis at 200 V for 35min
8 Pre-wet membrane in transfer buffer 10 minutes before use at room temperature
9 Cut the top right corner of a membrane and label the top left corner with the blot number
10 Prepare the transfer apparatus: fill the box half full with pre-cold transfer buffer Wet sponges and filter paper in transfer buffer
11 Carefully transfer the gel to the filter paper, such that the top right corner is on the right and faces away from the hinge
To ensure proper protein transfer, carefully position the membrane over the gel, aligning their nicked corners This step guarantees that proteins move from left to right on the membrane, with the marker on the left and samples numbered sequentially upwards Accurate placement is essential for reliable transfer and subsequent analysis.
Ensure the membrane and gel stay moist by keeping them wet and carefully eliminating any bubbles between them Complete the transfer process by layering filter paper and sponge to form a transfer sandwich, then securely clamp the tray Finally, close the transfer box and place it in an ice-filled container to maintain optimal conditions during transfer.
15 Perform blocking with PBST/5% non-fat dry milk and incubate for 2h, shaking at room temperature
16 Incubate with primary antibody overnight
17 Wash the membrane 3 times with 1×PBST, 5 min each time
18 Incubate the membrane scond Antibody Peroxidase Conjugated (1:2000 in PBST/5% non-fat dry milk, v/v) for 1 hour
19 Wash the cells 3 times with PBST, 5 min each
20 Mix the two substrate components at a 1:1 ratio to prepare the substrate Working Solution and incubate membrane 1 minute in the prepared Super Signal West Substrate Working Solution
21 Analyze the membrane and take images
1 Rinse cells briefly twice in 0.5ml/well ice-cold PBS w/o Ca 2+ /Mg 2+ at room temperature
2 Fix the samples in freshly made 0.5ml/well 4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature with shaking
3 Wash the samples three times with 0.5ml/well ice-cold PBS
Note: The cells can be stored in 0.02% (w/v) sodium azide in PBS at 4°C for several days
4 Incubate the samples for 10 min with 0.5ml/well PBS w/o Ca 2+ /Mg 2+ containing 0.5% Triton X-100 (in room temperature) with shaking
5 Wash cells in 0.5ml/well PBS three times, each for 5 min with shaking
Incubate the cells with 0.5 ml per well of blocking buffer containing 5% sheep serum, 5% donkey serum, 0.05% Tween-20, and 0.1% Triton X-100 in PBS for 1 hour to effectively block nonspecific antibody binding This step should be performed with gentle shaking to ensure optimal coverage and reduce background noise, utilizing serum from the species in which the secondary antibody was raised to enhance specificity.
7 Incubate cells in 150àl/well mixture of two primary antibodies in blocking buffer overnight at 4 o C with shaking
8 Decant the mixture solution and wash the cells three times in 0.5ml/well wash buffer, each for 5 min with shaking
9 Dilute the fluorophore-conjugated secondary antibody/antibodies, away from light, in blocking buffer Be sure that the correct isotype-specific secondary antibody for each primary antibody is used
Incubate the cells with 150 µL per well of a mixture of two secondary antibodies, each raised in different species and conjugated to distinct fluorochromes—FITC-conjugated sheep anti-mouse and TRITC-conjugated donkey anti-rabbit—in blocking buffer This step should be performed for 1 hour at room temperature in the dark with gentle shaking to ensure optimal binding and minimize background fluorescence.
11 Decant the mixture of the secondary antibody solution and wash three times with PBS each for 5 min in dark with shaking
12 4 drops of VECTASHIELD Mounting Medium with DAPI were added to each well and incubate for 1 minute
13 Visualize the cells using a fluorescence microscope equipped with the appropriate filters for different dyes and take images
1 Detach IMR 90 cells by dispase and collect them in the 1.5 mL tube
2 Wash the cells by CMRL-BSA medium
3 Count cell numbers and add 70,000 cells in each tube with 1 ml CMRL-BSA medium
4 Add 10 àl integrin antibodies to each tube
5 Seeding on the plates and incubate at 37 o C in CO2 incubator After incubation until the cells attach to the plates
6 Wash cells by CMRL-BSA medium for 3 times
7 Fix by 0.5 ml/well100% ethanol for 5 minutes
8 Stain the cells by 0.5 ml/well 0.4% crystal violet in methanol for 5 minutes
9 Wash the wells by dd H2O twice
10 Take Images count the colony numbers
5.5 Purification of total RNA from animal cells using spin technology
1 Carefully remove all medium by aspiration and wash twice by DPBS
2 Cells lysed directly by adding 600 àL Buffer RLT
3 Use pipet to mix and detach the cells and transfer to a new tube
4 Pass the lysate at least 5 times through a blunt 20G1 1 /2 needle fitted to an RNase-free syringe
5 Add 1 volume of 70% ethanol to the homogenized lysate, and mix well by pipetting
Transfer up to 700 μl of your sample, including any precipitate, to an RNeasy spin column placed in a 2 ml collection tube Gently close the lid and centrifuge for 15 seconds at ≥8000 x g (10,000 rpm), then discard the flow-through.
7 Add 700 μl Buffer RW1 to the RNeasy spin column Close the lid gently, and centrifuge for 15 s at ≥8000 x g (10,000 rpm) to wash the spin column membrane Discard the flow- through
8 Add 500 μl Buffer RPE to the RNeasy spin column Close the lid gently, and centrifuge for 15 s at ≥8000 x g (10,000 rpm) to wash the spin column membrane Discard the flow- through
9 Add 500 μl Buffer RPE to the RNeasy spin column Close the lid gently, and centrifuge for 2 min at ≥8000 x g (10,000 rpm) to wash the spin column membrane
Place the RNeasy spin column into a new 1.5 ml collection tube, then add 30–50 μl of RNase-free water directly onto the membrane to elute the RNA Gently close the lid and centrifuge for 1 minute at ≥8000 x g (10,000 rpm) to effectively recover the purified RNA.
5.6 Quantitative real time–polymerase chain reaction
Prepare the Thaw 2x QuantiTect Multiplex RT-PCR NoROX Master Mix, template RNA, primer and probe solutions, and RNase-free water, then mix the individual components on ice Always retrieve the QuantiTect Multiplex RT Mix from –20°C immediately prior to use, keep it on ice during the process, and promptly return any unused portions to –20°C storage afterward to ensure optimal stability and performance.
2 Prepare a reaction mix according to Table A1 (multiplex RT-PCR using the LightCycler 2.0)
Table A1 Reaction setup for duplex on RT-PCR for other cyclers
3 Mix the reaction mix thoroughly, and dispense appropriate volumes into PCR tubes, PCR capillaries, or the wells of a PCR plate
4 Add template RNA to the individual PCR tubes, capillaries, or wells
5 Program the real-time cycler according to Table A2.