Patent Application
20250051718
The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 1, 2020, is named 47460-108_ST25.txt and is 16,452 bytes in size.
A cell culture medium optimized in constituents and concentrations necessary to maintain pluripotent cellular state and cell proliferation, particularly of human induced pluripotent stem cells (hiPSCs), is provided.
Human induced pluripotent stem cells (hiPSCs) are functionally immortal and can proliferate without limit while maintaining the potential to differentiate to, hypothetically, all ˜220 cell lineages within the human body. hiPSC generation has become routine due to the simplicity of amplification of CD71+ blood proerythroblasts (Chou et al., 2015; Chou et al., 2011; Tan et al., 2014) or myeloid cells (Eminli et al., 2009; Staerk et al., 2010) and commercial Sendai virus-based reprogramming factor expression (Fujie et al., 2014; Fusaki et al., 2009). This simplicity has resulted in increased enthusiasm for the potential applications of hiPSC-derived cells across many fields, including regenerative medicine, disease modeling, drug discovery, and pharmacogenomics.
However, these applications require the culture of either large quantities of hiPSCs or hiPSC lines derived from large numbers of patients, and three major restrictions have become evident: 1, the cost of large-scale pluripotent cell culture, which is prohibitive for high patient-number projects; 2, the time-consuming requirement for daily media changes, which is particularly problematic for laboratories in industry; 3, inter-line variability in differentiation efficacy, which is highly dependent on pluripotent culture consistency and methodology.
Human induced pluripotent stem cell (hiPSC) culture has become routine, yet pluripotent cell media costs, frequent media changes, and reproducibility of differentiation have remained restrictive, limiting the potential for large-scale projects. Here, we describe the formulation of a novel hiPSC culture medium (B8) as a result of the exhaustive optimization of medium constituents and concentrations, establishing the necessity and relative contributions of each component to the pluripotent state and cell proliferation. B8 eliminates 97% of the costs of commercial media. The B8 formula is specifically optimized for fast growth and robustness at low seeding densities.
We demonstrated the derivation of 29 hiPSC lines in B8 as well as maintenance of pluripotency long-term, while conserving karyotype stability. This formula also allows a weekend-free feeding schedule without sacrificing growth rate or capacity for differentiation. Thus, this simple, cost-effective B8 media, will enable large hiPSC disease modeling projects such as those being performed in pharmacogenomics and large-scale cell production required for regenerative medicine. Human induced pluripotent stem cell (hiPSC) culture has become routine, yet pluripotent cell media costs, frequent media changes, and reproducibility of differentiation have remained restrictive, limiting the potential for large-scale projects. Here, we describe the formulation of a novel hiPSC culture medium (B8) as a result of the exhaustive optimization of medium constituents and concentrations, establishing the necessity and relative contributions of each component to the pluripotent state and cell proliferation.
Other methods, features and/or advantages is, or will become, apparent upon examination of the following figures and detailed description. It is intended that all such additional methods, features, and advantages be included within this description and be protected by the accompanying claims.
Demonstrated herein is a novel media formula (B8), thoroughly optimized to support high growth rate under low seeding density conditions, require minimal media exchanges, and at low cost, while maintaining differentiation reproducibility. This formula is capable of supporting both hiPSC generation and culture for >100 passages. Generation of B8 supplement aliquots suitable for making 100 liters of media is simple for any research lab with basic equipment, with complete bottles of media costing ˜$12 USD per liter.
A full protocol is provided, including detailed instructions for recombinant protein production in three simple steps. The protocol is made possible by the in-lab generation of three E. coli-expressed, codon-optimized recombinant proteins: an engineered form of fibroblast growth factor 2 (FGF2) with improved thermostability (FGF2-G3); transforming growth factor β3 (TGFβ3)—a more potent TGFβ able to be expressed in E. coli; and a derivative of neuregulin 1 (NRG1) containing the EGF-like domain. All plasmids for protein production are available through Addgene. With the commoditization of these protocols, we believe it is possible to substantially increase what is achievable with hiPSCs due to the near elimination of pluripotent cell culture costs and minimization of labor associated with cell culture.
Only five components were essential for hiPSC culture: insulin, sodium selenite, FGF2, DMEM/F12, (
A number of surprising results in development of the culture media. For example, neither a positive or negative effect of the addition of albumin (
A major issue with some commercial media is that although a weekend-free schedule is feasible, growth of hiPSCs is considerably slower and it is recommended to grow cells as low-density colonies. These low-density colonies are not compatible with subsequent monolayer differentiation protocols, as have become commonplace with the majority of lineages. The optimization of the B8 culture media specifically for fast monolayer growth, along with the incorporation of thermostable FGF2-G3, overcomes many of these issues while maintaining compatibility with common differentiation protocols.
The growth factors FGF2 and TGFβ1 represented more than 80% of the total medium costs. Optimization of the plasmids and generation of thioredoxin fusion proteins where necessary eliminates much of the complexity associated with inclusion bodies and the resulting refolding processes otherwise required. A typical 1 liter E. coli culture, which requires two days and basic laboratory skills, will usually provide 80 mg of FGF2-G3, enough for 800 liters of B8. Similarly, a 500 ml culture of TGFβ3 or NRG1 will commonly provide enough protein years of work (˜800,000 liters of B8 media). Additionally, the concentrations of these components can be reduced by 75% without a substantial impact on growth rate (both at 5 μg ml−1) (
In particular, the cell medium comprises, for growth of human induced pluripotent stem cells, a cell culture base medium; a Fibroblast Growth Factor 2; insulin; and a source of selenium. The cell culture medium is not required to contain Transforming Growth Factor beta 1 (TGFβ1), Activin A, or albumin. That is the cell culture medium may contain substantially no Transforming Growth Factor beta 1 (TGFβ1), Activin A, or albumin. The cell culture medium may contain trace amounts of Transforming Growth Factor beta 1 (TGFβ1), Activin A, or albumin. The cell culture medium may contain measurable amounts of Transforming Growth Factor beta 1 (TGFβ1), Activin A, or albumin, but not in a concentration sufficient to influence cell growth, differentiation or health.
In some aspects, insulin or IGF 1 may be used in the culture media. Recombinant forms of insulin, IGF1, any derivatives or variants that are cost effective to produce without any detriment to function may be substituted for insulin or IGF1. Similarly, a source of selenium may comprise a selenium salt, L-selenomethionine, selenocysteine, methylselenocysteine or similar compounds.
In some aspects, the cell culture medium may also contain TGFβ3, NRG1; transferrin, ascorbic acid, or a combination thereof. The cell culture medium may also contain thiazovivin. The cell culture medium may be characterized by a pH of 7.1 or by an osmolarity of 310 mOsm/l. The cell culture medium may also be characterized by sodium bicarbonate in an amount of 2438 μg/ml.
In some aspects the cell culture base medium is DMEM/F12. In some aspects the FGF2 is a recombinant protein defined as either SEQ ID NO: 4, 5, or 15, or a mixture thereof. In some aspects the FGF2 is a recombinant protein FGF2-G3 (SEQ ID NO: 15). In some aspects the selenite salt is sodium selenite. In some aspects, the TGFβ3 is a recombinant protein of SEQ ID NO: 16, NRG1 is a recombinant protein of SEQ ID NO: 17. In some aspects, the cell culture medium comprises: 40 ng/ml FGF2-G3, 20 μg/ml insulin, 20 ng/ml sodium selenite, formulated in a DMEM/F12 culture medium. As an alternative, the cell culture medium may include 40 ng/ml FGF2-G3 (SEQ ID NO: 15), 20μg/ml insulin, 20 ng/ml sodium selenite, 20 μg/ml transferrin, 0.1 ng/ml TGFβ3 (SEQ ID NO: 16), 0.1 ng/ml NRG1 (SEQ ID NO: 17), 200 μg/ml ascorbic acid 2-phosphate, 2438 μg/ml sodium bicarbonate formulated in a DMEM/F12 culture medium.
Also provided herein is a kit for preparation of a cell culture medium, the kit comprising: plasmids encoding FGF2-G3, TGFβ3, and NRG1; and instructions for preparing FGF2-G3, TGFβ3, and NRG1 protein and preparing a cell culture medium. The kit may further include culture medium, sodium selenite, insulin, transferrin, ascorbic acid 2-phosphate, sodium bicarbonate, or thiazovivin.
Also provided herein are methods of growing and passing human induced pluripotent stem cells (hiPSCs) in culture, the method comprising: obtaining a cell culture medium comprising: FGF2-G3 (SEQ ID NO: 15), insulin, sodium selenite, transferrin, TGFβ3 (SEQ ID NO: 16), NRG1 (SEQ ID NO: 17), ascorbic acid 2-phosphate, sodium bicarbonate formulated in a DMEM/F12 culture medium, preparing matrix coated plates; adding hiPSCs to the matrix, day 0; changing cell culture medium on day 1; passing cells on day 3.5 or growing cells for 7 consecutive days provided that at least one day of the 3.5 day passing or the 7-day cell growth cycle will not require changing the cell culture medium.
The culture media described herein suggest the use of a DMEM/F12 as a culture media base. However, any appropriate culture media base can be combined with the insulin, ascorbic acid, transferrin, selenite, FGF2, TGFβ, and NRG1as described herein. In fact, Chen et al. showed comparable results between DMEM/F12 and the comparatively simple MEMα. Any other basic defined culture media may also be used in combination with the insulin, ascorbic acid, transferrin, selenite, FGF2, TGFβ, and NRG1as described herein.
Media conditions required to culture human pluripotent stem cells has progressed steadily over the last 15 years, with significant breakthroughs coming from the discovery of the necessity for high concentrations of FGF2 (Xu et al., 2005), the use of TGFβ1 (Amit et al., 2004), and the elimination of knockout serum replacement (KSR) with the TeSR formula which contains 21 components (Ludwig and Thomson, 2007; Ludwig et al., 2006a; Ludwig et al., 2006b), followed by the first robust chemically defined formula, E8 (Beers et al., 2012; Chen et al., 2011) which consists of just 8 major components. A number of alternative non-chemically defined pluripotent formulations have been described including: CDM-BSA (Hannan et al., 2013; Vallier et al., 2005; Vallier et al., 2009), DC-HAIF (Singh et al., 2012; Wang et al., 2007), hESF9T (Furue et al., 2008; Yamasaki et al., 2014), FTDA (Breckwoldt et al., 2017; Frank et al., 2012; Piccini et al., 2015), and iDEAL (Marinho et al., 2015) (FIG. S1A).
Each of the available formulations consist of a core of three major signaling components: 1) insulin or IGF1 which bind INSR and IGF1R to signal the PI3K/AKT pathway promoting survival and growth; 2) FGF2 and/or NRG1 which bind FGFR1/FGFR4 or ERBB3/ERBB4 respectively, activating the PI3K/AKT/mTOR and MAPK/ERK pathways; and 3) TGFβ1, NODAL, or activin A which bind TGFBR1/2 and/or ACVR2A/2B/1B/1C to activate the TGFβ signaling pathway. NODAL is used less commonly in pluripotent media formulations due to the expression of NODAL antagonists LEFTY1/2 in human pluripotent stem cells (hPSC) (Besser, 2004; Sato et al., 2003) resulting in a requirement for high concentrations in vitro (Chen et al., 2011). In addition, numerous growth factor-free formulae utilizing small molecules to replace some or all growth factors in hPSC culture have been described (Burton et al., 2010; Desbordes et al., 2008; Kumagai et al., 2013; Tsutsui et al., 2011), however, these have not successfully translated to common usage. Recently, a growth factor-free formula AKIT was demonstrated (Yasuda et al., 2018), combining inhibitors of GSK3B (1-azakenpaulone), DYRK1 (ID-8), and calcineurin/NFAT (tacrolimus/FK506), albeit with much reduced proliferation and colony growth, as well as increased interline variability in growth. Finally, more than 15 commercial pluripotent media are also available in which the formulae are proprietary and not disclosed to researchers. These media represent the major cost for most hiPSC labs and considerably restrict research efforts. Some of these media formulae are suggested to support hiPSC growth without daily media changes or ‘weekend-free’, likely by using heparin sulfate to stabilize FGF2 that otherwise degrades quickly at 37° C. (Chen et al., 2012; Furue et al., 2008) and including bovine serum albumin (BSA) which acts as a multifaceted antioxidant.
Certain embodiments are described below in the form of examples. While the embodiments are described in considerable detail, it is not the intention to restrict or in any way limit the scope of the appended claims to such detail, or to any particular embodiment. The following Experimental Procedures are used throughout the Examples.
All pluripotent and reprogramming cell cultures were maintained at 37° C. in Heracell™ VIOS 160i direct heat humidified incubators (Thermo Scientific™) with 5% CO2 and 5% O2. Differentiation cultures were maintained at 5% CO2 and atmospheric (˜21%) O2. All cultures (pluripotent and differentiation) were maintained with 2 ml medium per 9.6 cm2 of surface area or equivalent. All media was used at 4° C. and was not warmed to 37° C. before adding to cells due to concerns of the thermostability of the FGF2 (Chen et al., 2012). We have found no detectable effects on cell growth from using cold media. All cultures were routinely tested for mycoplasma using a MycoAlert™ PLUS Kit (Lonza) and a 384-well Varioskan™ LUX (Thermo Scientific™) plate reader. E8 medium was made in-house as previously described (Burridge et al., 2015; Chen et al., 2011) and consisted of DMEM/F12 (Corning®, 10-092-CM), 20 μg ml−1 E. coli-derived recombinant human insulin (Gibco™, A11382IJ), 64 μg ml−1 L-ascorbic acid 2-phosphate trisodium salt (Wako, 321-44823), 10 μg ml−1 Oryza sativa-derived recombinant human transferrin (Optiferrin, In Vitria, 777TRF029-10G,), 14 ng ml−1 sodium selenite (Sigma, S5261), 100 ng ml−1 recombinant human FGF2-K128N (made in-house, see below), 2 ng ml−1 recombinant human TGFβ1 (112 amino acid, HEK293-derived, Peprotech, 100-21). Cells were routinely maintained in E8 medium on 1:800 diluted growth factor reduced Matrigel® (see below). E8 was supplemented with 10 μM Y27632 dihydrochloride (LC Labs, Y-5301), hereafter referred to as E8Y, for the first 24 h after passage. For standard culture, cells were passaged at a ratio of 1:20 every 4 days using 0.5 mM EDTA (Invitrogen UltraPure) in DPBS (without Ca2+ and Mg2+, Corning®), after achieving ˜70-80% confluence. Cell lines were used between passages 20 and 100. Other media components tested were: Human Long R3 IGF1 (Sigma, 91590C), thiazovivin (LC Labs, T-9753), recombinant human TGFβ3 (Cell Guidance Systems, GFH109), sodium bicarbonate (Sigma), NEAA (Gibco™), CD Lipids (Gibco™), fatty acid-free albumin (GenDEPOT, A0100). pH was adjusted with 10N HCl or 1N NaOH (both from Sigma) and measured using a SevenCompact™ pH meter (MettlerToledo). Osmolarity was adjusted with sodium chloride (Sigma) or cell culture water (Corning®) and measured with an osmometer (Advanced Instruments).
Our standard condition throughout was 2 ml of 1:800 reduced growth factor Matrigel® (Corning®, 354230) diluted in 2 ml of DMEM (Corning®, 10-017-CV) per well of 6-well plate or equivalent. Also tested were Geltrex® (Gibco™) and Cultrex® (Trevigen). Plates were made and kept in incubators at 37° C. for up to one month.
The full length (154 amino acid) FGF2 sequence (SEQ ID NO: 14) AAGSITTLP ALPEDGGSGA FPPGHFKDPK RLYCKNGGFF LRIHPDGRVD GVREKSDPHI KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LLASKCVTDE CFFFERLESN NYNTYRSKY TSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS with a K128N substitution (in bold/underlined) was codon optimized for E. coli with the addition of a BamHI site at the start (5′) of the sequence and an EcoRI site at the end (3′). This sequence was synthesized on a BioXp 3200 (Synthetic Genomics). The insert was then digested with BamHI and EcoRI (Anza, Invitrogen) and ligated with T4 DNA ligase (Anza) in to a pET-28a expression vector (Novagen/MilliporeSigma) and cloned in to One Shot™ BL21 Star™ (DE3) chemically competent E. coli (Invitrogen). E. coli were stored in 25% glycerol (Ultrapure, Invitrogen) at −80° C. A starter cultured was prepared by inoculating 10 ml of Terrific Broth (Fisher BioReagents) supplemented with 50 μg ml−1 kanamycin sulfate (Fisher BioReagents) in a bacterial tube (Corning® Falcon) and incubated in an Innova®−44 Incubator-Shaker (New Brunswick™) at 220 rpm overnight at 37° C. (for NRG1) or 30° C. (for FGF2 or TGFβ3). Protein expression was performed using a 2800 ml baffled shaker flask (BBV2800, Fisherbrand™) as follows: The whole 10 ml starter culture was added to 500 ml of MagicMedia™ (K6815, Invitrogen™), supplemented with 50 μg/ml kanamycin sulfate and incubated as above for 24 h at 37° C. (for NRG1) or 30° C. (for FGF2 or TGFβ3). The culture was harvested in to 2×250 ml centrifuge bottles (Nalgene®, 3120-0250) and centrifuged in an Optimia™ XPN-100 ultra centrifuge (Beckman Coulter) with a SW 32 Ti rotor at 5,000×g for 20 min at 4° C. Supernatant was carefully poured off and pellets were weighed and stored at −80° C. for downstream processing. Cells pellets were resuspended B-PER lysis buffer (Thermo Scientific™, 78248) using 5 ml of B-PER Complete Reagent per gram of bacterial cell pellet. Cells were incubated for 15 minutes at RT with gentle rocking. The bottles containing the lysates were then centrifuged in an ultracentrifuge at 16,000×g for 20 min at 4° C. Supernatants were collected and the cell debris was discarded. Purification was completed using a 3 ml HisPur™ Ni-NTA Spin Purification kit (Thermo Scientific™, 88229) following the manufacturers recommendations. To enhance the protein binding efficiency to the resin bed, the sample was incubated for 30 min at 4° C. Four elutes were collected, one every 10 min. The columns were reused following the manufacturer's regeneration protocol. The protein concentration was evaluated using Quant-iT™ Qubit® Protein Assay Kit (Invitrogen, Q3321) on a Qubit 3 fluorometer. The 6xHis tag was not cleaved as it has been previously demonstrated to not interfere with the FGF2 function (Soleyman et al., 2016). A standard 1 liter culture produced 80 mg of FGF2.
154 amino acid sequence (SEQ ID NO: 15): AAGSITTLP ALPEDGGSGA FPPGHFKDPK LLYCKNGGFF LRIHPDGR VD GTRDKSDPFI KLQLQAEERG VVSIKGVCAN RYLAMKEDGR LYAIKNVTDE CFFFERLEEN NYNTYRSRKY PSWYVALKRT GQYKLGSKTG PGQKAILFLP MSAKS with R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, and T121P substitutions (in bold) was codon optimized for E. coli was generated as above. FGF1-4X was similarly generated.
112 amino acid sequence (SEQ ID NO: 16): ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPCCVP QDLEPLTILY YVGRTPKVEQ LSNMVVKSCK CS was codon optimized for E. coli and generated as above, ligated in to a pET-32a expression vector, and cloned in to One Shot™ BL21 Star™ (DE3). The use of pET-32a results in the production of a thioredoxin-TGFβ3 fusion protein which prevents protein expression in inclusion bodies. It is not necessary to cleave the thioredoxin for TGFBB3 to be active. TGFβ1, TGFβ1m (C77S), and TGFβ3m (C77S) were similarly generated.
65 amino acid sequence (SEQ ID NO: 17): SHLVKCAEKE KTFCVNGGEC FMVKDLSNPS RYLCKCPNEF TGDRCQNYVM ASFYKHLGIE FMEAE, which is a truncated version of NRG1 containing just the EGF domain, was codon optimized for E. coli and generated as above, ligated in to a pET-32a expression vector, and cloned in to One Shot™ BL21 Star™ (DE3). The use of pET-32a results in the production of a thioredoxin-NRG1 fusion protein which prevents protein expression in inclusion bodies. It was found necessary to cleave the thioredoxin using Thrombin CleanCleave™ Kit (MilliporeSigma), followed by repurification, keeping the supernatant.
The hiPSC line 19-3 was dissociated with TrypLE (Gibco™, 12604-013) for 3 min at 37° C. and cells were resuspended in DMEM/F12, transferred to a 15 ml conical tube (Falcon) and centrifuged at 200×g for 3 min (Sorvall ST40). The pellet was resuspended in DMEM/F12, diluted to 1×105 cells per ml and 10,000 cells were plated per well in Matrigel® (1:800)-coated 12-well plates (Greiner) in the medium to be tested along with 2 μM thiazovivin for the first 24 h. Media were changed daily and cells were grown for 6 days. This lower than normal seeding density was used to allow the discovery of factors only detectable under more extreme conditions and therefore provide data on the robustness to the formulation.
Stock lysis buffer was prepared as 150 mM NaCl, 20 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100 and stored at 4° C. Fresh complete lysis buffer was prepared with final concentration of 1× Protease Inhibitor (Roche, 5892791001), 1× Phosphatase Inhibitor Cocktail 2 (P5726, Sigma), 1× Phosphatase Inhibitor Cocktail 3 (Sigma, P0044), 2 mM PMSF (Sigma, P7626), and 1% SDS Solution (Fisher Scientific, BP2436200). hiPSC were starved using B8 without FGF2 for 24 hours, then treated with media containing the corresponding FGF2 for 1 h. Media was then removed, and cells were washed once with DPBS, harvested with 0.5 mM EDTA in DPBS, and transferred into tubes. Samples were pelleted by centrifugation at 500×g for 3 minutes and the supernatant was discarded. The pellet was resuspend in 150 μl complete lysis buffer and incubated on ice for 30 min. Clear lysates were collected by centrifugation at 10,000×g for 10 min at 4° C. The protein concentration was measured with Qubit Protein Assay Kit (Invitrogen, Q33211) and Qubit 4 fluorometer. Lysates was stored in-80° C. before use. 10 μg of sample was prepared with NuPAGE™ LDS Sample Buffer (Invitrogen, B0007) and NuPAGE™ Reducing Agent (Invitrogen, B0009) according to the manufacturer's instructions and run on NuPAGE™ 10% Bis-Tris Gel (Invitrogen, NP0302BOX) and Mini Gel Tank system (Invitrogen, A25977) with Bolt MES SDS Running Buffer (Invitrogen, B000202) at 100 V for 1 h. SeeBlue Plus2 Pre-Stained Protein Standard (Invitrogen, LC5925) was used as a ladder. The gel was then transferred in Mini Trans-Blot Cell system (Bio-Rad, 1703930) on to a PVDF transfer membrane (Thermo Scientific™, 88518) at 240 mA for 1 h and 30 min. The membrane was blocked with 5% BSA (GenDEPOT, A0100) in 1% TBST (Fisher Scientific, BP2471-1, BP337-100) overnight. All the primary and secondary antibodies were diluted with 5% BSA in 1% TBST. Washes were done as a short rinse followed by 5 long washes for 5 min each. Both primary (Cell Signaling Technology, 9101, 9102) and secondary antibodies (92632211, LI-COR) were incubated for 1 h at RT. The blot was imaged with Odyssey CLx (LI-COR). The blot was stripped with Restore™ PLUS Western Blot Stripping Buffer (Thermo Scientific™, 46430) for 15 min at RT, rinsed with 1% TBST and reblocked with 5% BSA for 30 min.
Protocols were approved by the Northwestern University Institutional Review Boards. With informed written consent, ˜9 ml of peripheral blood was taken from each volunteer and stored at 4° C., samples were transferred to Leucosep tubes (Greiner) filled with Histopaque®-1077 (Sigma). 1×106 isolated peripheral blood mononuclear cells (PMBC) were grown in 24-well tissue culture-treated plates (Greiner) in 2 ml of SFEM II (Stem Cell Technologies) supplemented with 10 ng ml−1 IL3, 50 ng ml−1 SCF (KITLG), 40 ng ml−1 IGF1 (all Peprotech), 2 U ml−1 EPO (Calbiochem), 1 μM dexamethasone (Sigma) (Chou et al., 2015). 50% medium was changed every other day. After 12 days of growth, 6×104 cells were transferred to a well of a 24-well plate in 500 μL of SFEM II with growth factors supplemented with CytoTune™-iPS 2.0 Sendai Reprogramming Kit viral particle factors (Gibco™) (Fujie et al., 2014; Fusaki et al., 2009) diluted to 5% (1:20) of the manufacturer's recommendations. Cells were treated with 3.5 μL, 3.5 μl, and 2.2 μl of hKOS (0.85×108 CIU ml−1), hMYC (0.85×108 CIU ml−1), and hKLF4 (0.82×108 CIU ml−1), respectively at MOI of 5:5:3 (KOS:MYC:KLF4). 100% media was changed after 24 h by centrifugation (300×g, 4 min) to 2 ml fresh SFEM II with growth factors, and cells were transferred to one well of a 6-well plate (Greiner) coated with 1:800 Matrigel®. 50% medium was changed gently every other day. On day 8 after transduction, 100% of medium was changed to B8 medium. Medium was changed every day. At day 17 individual colonies were picked into a Matrigel®-coated 12-well plate (one colony per well).
hiPSCs were dissociated with TrypLE™ Express (Gibco™) for 3 min at 37° C. and 1×106 cells were transferred to flow cytometry tubes (Falcon, 352008). Cells were stained in 0.5% fatty acid-free albumin in DPBS using 1:20 mouse IgG3 SSEA4-488 clone MC-813-70 (R&D Systems, FAB1435F, lot. YKM0409121) and 1:20 mouse IgM TRA-1-60-488 clone TRA-1-60 (BD Biosciences, 560173, lot. 5261629) for 30 min on ice then washed. Isotype controls mouse IgG3-488 clone J606 (BD Biosciences, 563636, lot. 7128849) and mouse IgM-488 clone G155-228 (BD Bioscience, 562409, lot. 7128848) were used to establish gating. All cells were analyzed using a CytoFLEX (Beckman Coulter) with CytExpert 2.2 software.
hiPSCs were dissociated with 0.5 mM EDTA and plated onto Matrigel®-treated Nunc Lab-Tek II 8-chamber slides in B8 medium for three days (B8T for the first 24 h). Cells were fixed, permeabilized, and stained for OCT4, SSEA4, SOX2, TRA-1-60 with PSC 4-Marker Immunocytochemistry Kit (Life Technologies, A24881, Lot. 1610720) according to the manufacturer's instructions. Cells were washed three times and mounted with ProLong™ Diamond Antifade Mountant with DAPI (Invitrogen). Slides were imaged with a Ti-E inverted fluorescent microscope (Nikon Instruments) and a Zyla sCMOS camera (Andor) using NIS-Elements 4.4 Advanced software.
Population doubling level (PDL) was calculated according to the following formula:
Where n=cell number and n0=number of cells seeded
Differentiation into cardiomyocytes was performed according to previously described protocol with slight modifications (Burridge et al., 2015; Burridge et al., 2014). Briefly, hiPSCs were split at 1:20 ratios using 0.5 mM EDTA as above and grown in B8 medium for 4 days reaching ˜75% confluence. At the start of differentiation (day 0), B8 medium was changed to CDM3 (chemically defined medium, three components) (Burridge et al., 2014), consisting of RPMI 1640 (Corning®, 10-040-CM), 500 μg ml−1 fatty acid-free bovine serum albumin (GenDEPOT), and 200 μg ml−1 L-ascorbic acid 2-phosphate (Wako). For the first 24 h, CDM3 medium was supplemented with 6 μM of glycogen synthase kinase 3-β inhibitor CHIR99021 (LC Labs, C-6556). On day 1, medium was changed to CDM3 and on day 2 medium was changed to CDM3 supplemented with 2 μM of the Wnt inhibitor Wnt-C59 (Biorbyt, orb181132). Medium was then changed on day 4 and every other day for CDM3. Contracting cells were noted from day 7. On day 14 of differentiation, cardiomyocytes were dissociated using DPBS for 20 min at 37° C. followed by 1:200 Liberase TH (Roche) diluted in DPBS for 20 min at 37° C., centrifuged at 300 g for 5 min, and filtered through a 100 μm cell strainer (Falcon).
hiPSCs were grown to approximately 50-70% confluent and differentiated according to an adapted version of a protocol previously described (Patsch et al., 2015). On day 5 of differentiation, endothelial cells were dissociated with Accutase® (Gibco™) for 5 min at 37° C., centrifuged at 300 g for 5 min, and analyzed.
hiPSCs were split at 1:20 ratios using 0.5 mM EDTA as above and grown in B8T medium for 1 day reaching ˜15% confluence at the start of differentiation. Surface ectoderm differentiation was performed according to an adapted version of previously described protocols (Li et al., 2015; Qu et al., 2016). On day 4 of differentiation, epithelial cells were dissociated with Accutase (Gibco™) for 5 min at 37° C., centrifuged at 300 g for 5 min, and analyzed.
Cardiomyocytes were dissociated with Liberase TH as described above, transferred to flow cytometry tubes and fixed with 4% PFA (Electron Microscopy Services) for 15 min at RT, and then permeabilized with 0.1% Triton X-100 (Fisher BioReagents) for 15 min at RT, washed once with DPBS, and stained using 1:33 mouse monoclonal IgG1 TNNT2-647 clone 13-11 (BD Biosciences, 565744, lot. 7248637) for 30 min at RT and washed again. Isotype control mouse IgG1-647 clone MOPC-21 (BD Biosciences, 565571, lot. 8107668) was used to establish gating. Endothelial cells were dissociated with Accutase® as described above, transferred to flow cytometry tubes and stained with 1:100 mouse IgG2a CD31-647 clone M89D3 (BD Bioscience, 558094, lot. 8145771) for 30 min on ice then washed once with DPBS. Isotype control mouse IgG1-647 clone MOPC-21 (BD Biosciences, 565571, lot. 8107668) was used to establish gating. Epithelial cells were dissociated with Accutase® as described above, transferred to flow cytometry tubes, fixed with 4% PFA (Electron Microscopy Services) for 10 min at RT, and then permeabilized with 0.1% saponin (Sigma) in DPBS for 15 min at RT. Cells were washed once in wash buffer (DPBS with 5% FBS, 0.1% NaN3, 0.1% saponin), stained with 1:200 mouse IgG1 KRT18-647 clone DA-7 (BioLegend, 628404, lot. B208126) for 30 min at RT, then washed twice with wash buffer. Isotype control mouse IgG1-647 clone MOPC-21 (BD Biosciences, 565571, lot. 8107668) was used to establish gating. All cells were analyzed using a CytoFLEX (Beckman Coulter) with CytExpert 2.2 software.
Data were analyzed in Excel or R and graphed in GraphPad Prism 8. Detailed statistical information is included in the corresponding figure legends. Data were presented as mean±SEM. Comparisons were conducted via an unpaired two-tailed Student's t-test with significant differences defined as P<0.05 (*), P<0.01 (**), P<0.005 (***), and P<0.0001 (****). No statistical methods were used to predetermine sample size. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.
As a first step, concentrations of matrices on which hiPSCs are grown was determined. Although laminin-511 (Rodin et al., 2010), laminin-521 (Rodin et al., 2014), vitronectin (Braam et al., 2008), and Synthemax™-II (Melkoumian et al., 2010) are suitable for hiPSC culture (Burridge et al., 2014), none are appropriately cost-effective, and in the case of vitronectin or Synthemax-II, also not suitable for subsequent differentiation (Burridge et al., 2014). Matrigel®, although an undefined product (Hughes et al., 2010), is a cost-effective and commonly used matrix with substantial data using it at 50 μg cm−2 (Ludwig et al., 2006a). Comparing two similar commercial products, Matrigel® (Corning®) and Cultrex®/Geltrex® (Trevigen/Gibco™M), we found that both be used at concentrations as low as 10 μg cm−2 (a 1:1000dilution) (
A pluripotent growth assay modeled on that used by Ludwig et al. and Chen et al. was established after determining that neither automated cell counting of dissociated cells (6-well) or small format survival assays (i.e. 96-well) were suitably robust. The growth assay may be outlined as follows:
During the development of this assay platform, we found that precise measurement of relative growth was a suitable surrogate for metrics of the pluripotent state (such as NANOG expression). When cells began to spontaneously differentiate, such as when TGFβ1 is omitted from the formula, the resulting slowing of growth was easily detectable.
Each component of a commercially available cell culture media was evaluated for performance and cost effectiveness. The commercially available cell culture media comprises:
A range of concentrations for each of the six major hiPSC medium components (
Referring now to
The addition of 2 μM thiazovivin for the first 24 h marginally, though not significantly, improved growth over 10 μM Y27632 and was ˜5× more cost-effective choice (
Some recent hiPSC growth formulae have suggested that the addition of high levels (2×) of non-essential amino acids (NEAA) and/or low levels (0.1×) of chemically defined lipids enhance growth (
Supplementation with bovine serum albumin (BSA), a common hPSC media component, did not have positive or negative effects on growth and was excluded to maintain a chemically defined formula (
The DMEM/F12 basal media of Corning® contains higher levels of sodium bicarbonate (˜29 mM or 2438 mg 1−1 ) compared to DMEM/F12 from other manufacturers. Supplementation of Gibco™ DMEM/F12, which contains 14 mM of sodium bicarbonate, with 20 mM of additional sodium bicarbonate has recently been demonstrated to be advantageous to hiPSC growth rate by suppressing acidosis of the medium (Liu et al., 2018). However, the standard 29 mM of sodium bicarbonate was optimal according to
The effect of pH and osmolarity on growth were also evaluated. A pH 7.1 (
In initial studies, a commercial recombinant human FGF2 was provided. This constituent accounted for >60% of the media cost. Additional FGF2 variants were then assessed. A FGF2 sequence with E. coli-optimized codon usage to enhance yield and a K128N point mutation to improve thermostability (Chen et al., 2012) was inserted into a recombinant protein production plasmid (pET-28a) utilizing dual 6xHis tags that were not cleaved during purification (
Our initial short-term experiments provided preliminary optimizations, yet we were aware from previous experiments that some variables, such as the elimination of TGFβ1, had minimal effects short-term and would only have detectable negative effects in long-term experiments. Thus the effectiveness of a long-term assay was evaluated with the following assay protocol:
Each experiment was independently repeated at least 5 times. These experiments again confirmed concentrations of insulin (20 μg ml−1;
As TGFβ1 is now the costliest component (˜20% of total cost) we found that this could be used at lower concentrations than previously suggested (0.1 ng ml−1) even in a long-term assay (
Finally, two hESC media formulations that we studied, DC-HAIF and iDEAL, contain both FGF2 and neuregulin 1 (NRG1). We found that supplementation with all tested levels of NRG1 enhanced growth by >15% over FGF2-G3 alone (
One final B8 media formulation of the invention derived from these long-term assay optimizations is:
The suitability of the B9 media to generate hiPSC lines was confirmed. We generated hiPSC lines from 26 patients using established protocols but using B8. Lines were successfully generated from all donors and passed standard assays for pluripotency including flow cytometry for SSEA4 and TRA-1-60 (
With the understanding that B8 supports hiPSC growth across a variety of sub-optimal conditions (
As will culture of cells in any media for an extended period of time, other factors should be considered. For example, we know that the L-glutamine in the medium is unstable at 37° C. and that the concentration is reduced by about a third over four days. We also know that cells are producing ammonia, reducing the pH of the media, although this buffered partially by the HEPES and sodium bicarbonate. Finally, hiPSCs release autocrine or paracrine factors into the media that may induce differentiation and the increase in these factors over time has not been decoupled from the use of nutrients and production of metabolic waste.
“About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.
The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of and “consisting of” those certain elements. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).
As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. See In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term “consisting essentially of” should not be interpreted as equivalent to “comprising.” Moreover, the present disclosure contemplates that in some embodiments, any feature or combination of features can be excluded or omitted.
To illustrate, if the specification states that a complex comprises components A, B, and C, it is specifically intended that any of A, B, or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise stated, and each separate value is incorporated into the specification as if it were individually recited. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Unless otherwise defined, all technical terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
As stated above, while the present application has been illustrated by the description of embodiments, and while the embodiments have been described in considerable detail, it is not the intention to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art, having the benefit of this application. Therefore, the application, in its broader aspects, is not limited to the specific details and illustrative examples shown. Departures may be made from such details and examples without departing from the spirit or scope of the general inventive concept.
This application claims benefit of U.S. Provisional Application No. 62/902,561, filed Sep. 19, 2019, which is incorporated herein by reference in its entirety.
This invention was made with government support under contract HL 121177 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62902561 | Sep 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17025953 | Sep 2020 | US |
| Child | 18929526 | US |
