Editor’s Choice Article 23 August 2023
1 Department of Biomarkers and Investigative Pathology Unit (BIPU), Charles River Laboratories, Mattawan, MI 49071, USA
2 Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA
3 Department of Chemistry, Oakland University, Rochester, MI 48309, USA
4 Department of Bioengineering, Oakland University, Rochester, MI 48309, USA
Human embryonic stem cells and induced pluripotent stem cells (hPSC) have an unprecedented opportunity to revolutionize the fields of developmental biology as well as tissue engineering and regenerative medicine. However, their applications have been significantly limited by the lack of chemically defined and xeno-free culture conditions. The demand for the high-quality and scaled-up production of cells for use in both research and clinical studies underscores the need to develop tools that will simplify the in vitro culture process while reducing the variables. Here, we describe a systematic study to identify the optimal conditions for the initial cell attachment of hPSC to tissue culture dishes grafted with polymers of N-(3-Sulfopropyl)-N-Methacryloxyethyl-N, N-Dimethylammoniun Betaine (PMEDSAH) in combination with chemically defined and xeno-free culture media. After testing multiple supplements and chemicals, we identified that pre-conditioning of PMEDSAH grafted plates with 10% human serum (HS) supported the initial cell attachment, which allowed for the long-term culture and maintenance of hPSC compared to cells cultured on Matrigel-coated plates. Using this culture condition, a 2.1-fold increase in the expansion of hPSC was observed without chromosomal abnormalities. Furthermore, this culture condition supported a higher reprogramming efficiency (0.37% vs. 0.22%; p< 0.0068) of somatic cells into induced pluripotent stem cells compared to the non-defined culture conditions. This defined and xeno-free hPSC culture condition may be used in obtaining the large populations of hPSC and patient-derived iPSC required for many applications in regenerative and translational medicine.
Human pluripotent stem cells (hPSC): human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), have the unique abilities to replenish themselves through self-renewal and to differentiate into different types of specialized cells [1,2,3]. Since the first report of the derivation of hESCs in 1998 [2] and the successful development of hiPSCs from somatic cells in 2007 [1,3], stem cell research is rapidly progressing from basic research to the clinical arena with the hope that derivatives of these cells can be used to treat and manage debilitating diseases [4,5]. These cells also play essential roles in the study of organogenesis and tissue regeneration and represent an important cell source for cell-based therapies and drug development. Continued growth in cell therapies has created a consensus for the need for specialized and defined materials to translate stem cell studies into clinical trials.
Many of the current culture conditions are still based on undefined support systems that contain xenogeneic components, limiting the ability to use hPSC-derived cells to treat debilitating diseases [6,7,8]. In addition to the risk of transmitting pathogens and the introduction of tumor-derived growth factors, culture conditions that depend on undefined support systems affect experimental reproducibility and the ability to interpret mechanistic studies due to lot-to-lot variations and undefined conditions. This ultimately hinders the transition of hPSC-derived products into a clinical setting.
To overcome these problems, a significant effort has been made to understand the signaling pathways and molecules required to maintain the self-renewal and pluripotent state of hPSCs, resulting in the formulation of defined and xeno-free culture media. However, as anchorage-dependent cells, hPSCs also require specific extracellular matrices (ECM) as substrates to maintain their self-renewal [8]. Hence, the optimization of culture systems for hPSCs is not limited to defining the culture medium components, but also includes the substrates and environmental cues, among many other factors. To address these issues, natural or recombinant ECM proteins have been used to maintain hPSC self-renewal [9,10,11]. However, not all ECM proteins are suitable for hPSC culture as they cannot maintain an undifferentiated hPSC population or allow the clonal survival of hPSCs without the use of apoptosis inhibitors. The high production costs, labor-intensive cell passaging, and the limited scale-up potential associated with many of these ECM substrates have driven researchers to seek alternative substrates in the synthetic polymer arena composed of RGD (Arg-Gly-Asp) peptides covalently immobilized on an acrylate coating [12,13,14,15,16]. We reported the first fully defined synthetic polymer coating, which maintained the long-term growth, self-renewal, and pluripotency of hPSCs in a human cell conditioned medium (CM) and a defined serum-free medium, using the grafting of poly [2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) on tissue culture polystyrene (TCP) dishes [17]. Here, we report an optimization in the protocol through the use of PMEDSAH-grafted (PMEDSAH-g) TCP dishes on the culture and derivation of hPSC, which is compatible with multiple chemically defined and xeno-free culture medium formulations that exist in the market. We also show the successful derivation and long-term culture of hiPSC under defined and xeno-free conditions.
The following chemicals and materials were used: Angiopoietin-1 (ang1)-based peptide QHREDGS, retinoic acid (RA) (ATRA), integrin-activating antibody, calcium chloride dehydrates, magnesium chloride hexahydrate, manganese (II) chloride tetrahydrate, phorbol 12-myristate 13-acetate (PMA), and human serum (HS) were purchased from Sigma-Aldrich (Sigma-Aldrich Corp., Milwaukee, WI, USA). Fetal bovine serum (FBS), Teflon-coated magnetic 1″ stir bar, and stir plate were purchased from VWR (VWR International, Radnor, PA, USA). Knockout serum replacement (KOSR), anhydrous ethanol, and sodium chloride were obtained from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). The monomer N-(3-Sulfopropyl)-N-Methacryloxyethyl-N, N-Dimethylammoniun Betaine was purchased from Monomer-Polymer and Dajac labs (Monomer-Polymer and Dajac labs, Ambler, PA, USA). The TCPS of 35 mm in diameter were purchased from BD Falcon (Thomas Scientific, Swedesboro, NJ, USA). The UV Ozone cleaner (model no. 342) was purchased from Jelight Company (Jelight Company Inc., Irvine, CA, USA), and the transonic 52 ultrasonic cleaner (50/60 Hz) was obtained from Emerson (Emerson Electric, St. Louis, MO, USA). The cylindrical reaction vessel (500 mL), reaction vessel lid (3 × 24/40 necks), Viton O-ring, reactor clamp, support clamp for reaction vessel, vacuum/air manifold, condenser (24/40 Joint, 300-mm Jacket Length), heating mantle, heating mantle controller, and 24/40 glass stopper were purchased from Chemglass (Chemglass Life Science, Vineland, NJ, USA). The following xeno-free and chemically defined media were used for the hPSC culture: StemFlex (Gibco TM ThermoFisher Scientific, Waltham, MA, USA), mTeSR TM Plus (Stemcell TM Technologies, Vancouver, Canada), PluriSTEM TM (Sigma-Aldrich Corp.), and StemFit (Reprocell, Beltsville, MD, USA).
The UV ozone-initiated free radical polymerization was carried out in a fume hood with connections for nitrogen gas. Each reaction was used to prepare 24 TCP dishes, and this procedure was reproduced 10 times to produce 240 PMEDSAH-grafted plates. The monomer solution consisting of 0.25 M MEDSAH was dissolved in a mixture of deionized water and ethanol (4:1, v/v) in a 500 mL reaction vessel. The solution was degassed for 60 min through nitrogen purge. Then, the monomer solution was heated to 72 °C. While the reaction vessel was being heated, TCP dishes were activated through UV ozone treatment for 45 min to create initiation sites on the surface. After activation, the dishes were transferred to the reaction vessel and the temperature was raised to 76–80 °C. The surface-initiated polymerization occurred over a 2 h time period under a nitrogen atmosphere at 76–80 °C. Once the process was completed, grafted dishes were removed from the reaction vessel and rinsed with 1% saline (v/v) solution at 50 °C, followed by ultra-sonication in DI-water and air drying inside the hood.
Freshly prepared PMEDSAH-g dishes were treated with 10% HS (v/v DMEM/F12) for 30 min at room temperature, followed by a couple of washes with cold sterile Dulbecco’s phosphate buffered saline (D-PBS). Two sets of 10% HSt-PMEDSAH-g dishes were prepared. The first set, labeled as wet (W10% HSt-PMEDSAH-g), was used right after its preparation for culturing hPSC for 20 consecutive passages. For the other set of 10% HSt-PMEDSAH-g dishes, after their washes with cold sterile D-PBS, the dishes were air dried and wrapped with parafilm and stored at 4 °C until their use for cell culture, and were labeled as dry (D10% HSt-PMEDSAH-g). These plates were used for weekly passaging of hPSC for 13 consecutive passages. In both conditions, after every fifth passage, the cells were characterized for self-renewal and pluripotency.
Matrigel hESC-qualified (Corning, Glendale, AZ, USA) was diluted to a concentration of 0.1 mg/mL in cold Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12; GIBCO), which was then applied to TCP dishes. This coating was allowed to polymerize at room temperature during the 2 h incubation. Excess Matrigel-DMEM/F12 solution was aspirated, and the dishes were washed with cold sterile D-PBS followed by seeding of the cells.
NIH-approved hESC lines H1 and H9 (WiCell Research Institute, Madison, WI, and three hiPSC derived in our laboratory [18] (hGF2-iPSCs, hGF4-iPSCs, hFF [human foreskin fibroblasts] iPSC) were cultured on 10% HSt-PMEDSAH and Matrigel-coated dishes at 37 °C with 5% CO 2 using chemically defined media. Colony growth and development were observed every 48 h and the differentiated cells were mechanically removed using a sterile pulled-glass pipet under a stereomicroscope (LeicaMZ9.5, Leica Microsystems Inc., Buffalo Grove, IL, USA). Undifferentiated colonies were passaged weekly using L7-hPSC passaging solution (Lonza) and with supplementation of 10 μ m of ROCK inhibitor (Sigma) [19] at the freshly passaged cells. After dissociation into single cells, cell counting was performed, and 10,000 cells (1000 cells/cm 2) were plated on 10% HSt-PMEDSAH-g dishes and Matrigel-coated dishes and cultured for 7 days. The chemically defined culture medium was replaced every other day. Transmitted light images of cells and colonies were taken using an EVOS®FL Cell Imaging System (Thermo Fisher Scientific).
During 5 initial and consecutive weekly passages, the total number of cells grown on each plate of 10% HSt-PMEDSAH and Matrigel was counted and recorded at the time of passage for comparative analysis. From the total number of cells obtained, 10,000 cells were used per passage into a new substrate. A theoretical yield of the total cell number of hPSC obtained on different substrates was calculated assuming that all cells would be passaged each week instead of the only 10,000 single cells that were seeded. The theoretical yield of cells was determined with the formula CN(n+1) = CN n × TN(n+1)/10,000, in which CN is the calculated total cell number, TN is the total cell number and n is the passage number.
Cells grown on 10% HSt-PMEDSAH-g dishes were fixed in 4% paraformaldehyde for 10 min at room temperature followed by permeabilized with 0.1% Triton X-100 for 10 min. Primary antibodies raised against OCT3/4 (Cell signaling), SOX2 (MilliporeSigma, Burlin-tong, MA, USA), NANOG (Abcam, Cambridge, UK), TRA-1-60 (Abcam), and TRA-1-81 (MilliporeSigma) were diluted in 1% normal donkey serum and incubated overnight at 4 °C with gentle shaking and detected with respective secondary antibodies. Micrographs were captured using an EVOS®FL Cell Imaging System (Thermo Fisher Scientific).
Cells were washed with D-PBS and harvested through incubation in L7-hPSC passaging solution. After this, cells were incubated for 30 min in dark at 4 °C, first with human IgG to block un-specific binding and then with human/mouse NANOG APC-conjugated antibody (Biolegend, San Diego, CA, USA), OCT4 PE-conjugated antibody (Biolegend), SSEA3 APC-conjugated antibody (R&D systems), SSEA4 PE-conjugated antibody (R&D systems, Minneapolis, MN, USA), TRA-1-60 FITC-conjugated antibody (Biolegend), and TRA-1-81 PE-conjugated antibody (Biolegend). At least 10,000 events were acquired for each sample using the BD FACSCanto II and BD FACSAria (BD Biosciences) instruments and the flow cytometry data were analyzed using the FlowJo software.
RNA extraction from cells was conducted by directly adding Trizol (Thermo Scientific) into the culture dishes. RNA was isolated and purified using the Direct-zol™ RNA Miniprep (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocol. RNA quality and concentration were obtained using a NanoDrop™ 2000c Spectrophotometer (Thermo Scientific). Reverse transcription from 1 µg of total RNA into cDNA was performed using SuperScript™ VILO™ cDNA Synthesis Kit (Invitrogen™, Waltham, MA, USA). Quantitative PCR was then performed using TaqMan probes (Applied Biosystems, Waltham, MA, USA).
