Standardization of human-induced pluripotent stem cells by optimizing culture conditions and monitoring key parameters

doi:10.21203/rs.3.pex-2049/v1

Method Article

Standardization of human-induced pluripotent stem cells by optimizing culture conditions and monitoring key parameters

https://doi.org/10.21203/rs.3.pex-2049/v1

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The differentiation potential of human-induced pluripotent stem cells (hiPSCs) is not constant among clones, therefore standardization of iPSCs as a starting material is a key issue for the dissemination of hiPSC-based cell therapy. We showed that hiPSCs cultured with medium that supported the glycolytic pathway demonstrated a high level of chromodomain-helicase-DNA-binding protein 7 (CHD7) expression and retained differentiation potential. Notably, hiPSCs cultured with medium that supported mitochondrial activity expressed a low level of CHD7 and lost differentiation potential. In addition, we showed secretion of kynurenine by undifferentiated hiPSCs, and 2-AAA when differentiated. Thus, we propose re-cloning iPSC clones with medium supporting the glycolytic pathway for standardization, and measuring the expression of CHD7 by flow cytometry to check the differentiation potential and measuring Kynurenine and 2-AAA levels in the culture medium to monitor and assure the differentiation status of cells during culture.

Regenerative medicine (RM) is an emerging field of medicine with which intractable diseases are cured with cells, tissues, and organs manufactured. Human pluripotent stem cells (hPSCs) are an ideal source for RM products because hPSCs, in theory, undergo unlimited growth and differentiate into a variety of cell types. The first hPSCs, human embryonic stem cells, were established in 1998¹. Human-induced pluripotent stem cells (hiPSCs), which were first described in 2007² , have overcome ethical concerns in their derivation. The birth of iPSCs substantially increased the opportunities for RM because the technology has facilitated the establishment of hiPSCs from a variety of cells and tissues^2,3. Several hundred hiPSC clones have been established, which provide hope for the realization of hiPSC-based cell therapy⁴.

Nevertheless, there are some issues to resolve in the development of hPSC-based cell therapy. Specifically, the characteristic features of hPSCs differ, especially with respect to the differentiation potential or preference, even among hESCs, and the need to select a specific PSC clone, depending on the application or the target cells. Thus, we need to screen hPSCs for their differentiation potential and develop a tailor-made protocol for each clone. The differentiation bias of PSCs has been discussed since 2008⁵. The choice of hPSC line is crucial because a revision of the standard operating procedure (SOP), including material changes for commercial scale manufacturing will require additional time, costs, and workload. In contrast, there are limitations to the availability of clinical hPSC lines that meet Current Good Manufacturing Practice (CGMP) regulations⁶, as well as human leukocyte antigen (HLA) matching. Gene editing to knock out HLA I/II is another option by which HLA typing issues can be overcome^7,8, but CGMP regulatory requirements cannot be skipped. Genetic abnormalities that occur during prolonged culture periods are another concern. Genetic abnormalities include single nucleotide variations (SNVs) and copy number variations (CNVs), which occur incessantly and require another assessment to address permissible genetic abnormalities from a regulatory science perspective. We recently reported the correlation between genetic abnormalities in hiPSC derivatives and abnormal tissue formation⁹, and concluded that detection of > 3 CNVs, especially in 2 hot spots (14q32.33 and 17q12 loci), is a high predictive index for the occurrence of abnormal tissues or tumors. Therefore, in addition to a set of quality control (QC) tests to assess differentiation potential, speedy, cost-effective, and robust genetic tests are also essential as a safety test to commence PSC-derived cell therapy. Avoiding a prolonged culture period is critical to reducing the occurrence of genetic abnormalities.

It Is well-known that PSCs and cancer cells preferentially utilize the glycolytic pathway, rather than mitochondrial oxidative phosphorylation (OXPHOS) to generate the ATPs required for the biological activity of cells (the so-called Warburg effect)¹⁰. PSCs proliferate vigorously, thus maintaining self-renewal potential without further differentiation. We recently showed that the differentiation potential of PSCs has a positive relationship with the expression of chromodomain-helicase-DNA-binding protein 7 (CHD7)¹¹ and an inverse relationship with the activation of mitochondrial function. Therefore, cells cultured with medium that supports the glycolytic pathway, the activity of mitochondria in cells remains low, while they demonstrate differentiation potential with high expression level of CHD7. Cells cultured in medium preferentially supports the OXPHOS express low level of CHD7 and lost the differentiation potential manifested by no or few embryoid bodies (EB) formation in EB formation assay. Interestingly, genetic marking of cells in culture with their CVN profiles has revealed that cells cultured in medium preferentially support glycolytic pathway and the OXPHOS are distinct. That suggests that the medium selects the cells exhibiting a growth advantage under the given culture and these cells become dominant in the culture in a couple of passages, and does not support the idea that medium directly control the level of CHD7 expression¹². In addition, we showed that the differentiation process is coupled with upregulation of mitochondrial activity when cells differentiate from an undifferentiated state, and also the overexpression of CHD7 by introduction of mCHD7 induced the three germ layers differentiation simultaneously¹². Therefore, differentiation process of PSC is coupled with the activation of mitochondria and increased expression level of CHD7 respectively, suggesting that there is a counterbalance to maintain cells in an undifferentiated state. Because the culture medium primarily determines the level of mitochondrial activation, the maximum permissible level of CHD7 expression not to undergo differentiation spontaneously is determined secondarily by the medium formula^12,13. A study involving the introduction of siCHD7 also showed that CHD7 is positively correlated with the proliferation rate, and cells with higher CHD7 expression become dominant during culture and the expression level of CHD7 in cells converge at the highest permissible level¹². Therefore, improvement in the differentiation potential and homogeneity of cells in term of differentiation potential in culture can be evaluated by comparing the level of CHD7 expression (mean becomes higher) and convergence of CHD7 expression (CV) by flow cytometric analysis before and after culture medium changes respectively¹². This finding led to the notion of a novel protocol toward the standardization of PSCs while assuring differentiation potential by re-cloning already established PSCs through medium selection.

In addition to the culture medium, the other factor that affects the maintenance of self-renewal potential and differentiation potential of PSCs is the biophysical property of the extracellular matrix (ECM). hPSCs form monolayer colonies for growth in two-dimensional (2-D) cultures, and spontaneous differentiation inevitably occurs along the rim of colonies where cell-to-cell contact is lacking at the open side. This is in stark contrast to cells located in the middle of the colony that receive signals from cell-to-cell contact along cell membranes, seamlessly and evenly, thus allowing symmetric cell division and the generation of two identical daughter cells (self-renewal potential). Cells located along the rim of the colony undergo asymmetric division and generate two different daughter cells, thus interrupting self-renewal potential coupled with the initiation of differentiation. The initiation of differentiation is associated with the activation of the OXPHOS that generates the reactive oxygen species (ROS) as a result, and ROS can be detected by staining with superoxide dismutase 2 (SOD2) or direct production of ROS¹². The same event will occur in the cells located along the surface of spheres in 3-D culture lacking cell-to-cell contract in one open end. Because spontaneous differentiation is an inevitable event, it is important to determine how to minimize the contamination of differentiated cells in culture. To this end, we need to avoid ECM with potent adhesive properties, such as laminin 511 (L511) or L521 in the culture dish coating material, because differentiated cells have reduced adhesion properties compared to undifferentiated cells, and these differentiated cells cannot be removed effectively during passage if seeded on sticky L511 or L521¹². Truncated recombinant human vitronectin (rhVNT-N) with weak adhesive properties is recommended for use as a dish coating material in PSC cultures to effectively remove differentiated cells at the single cell level during cell passage¹². These findings are useful to set up the optimum culture protocol to maintain the purity of undifferentiated cells in culture and the size of the undifferentiated cell population during long-term culture.

Assurance of cell quality during mass culture of undifferentiated PSCs is another issue to be addressed from a quality control viewpoint that can be achieved by monitoring traceable metabolic biomarkers reflecting the differentiation status of PSCs during the culture. We have demonstrated tryptophan uptake and kynurenine secretion by hPSCs while cells are maintained in an undifferentiated state¹⁴. Kynurenine acts as autocrine factor that binds with an intracellular receptor (aryl hydrocarbon receptor [AhR]) and supports the expression of self-renewal genes¹⁴. In contrast, the kynurenine catabolic pathway is activated when hPSCs begin to differentiate, and 2-aminoadipic acid (2-AAA), an end-product of the pathway, can be detected in culture media as an early sign of differentiation. Kynurenine in culture medium is always detectable when PSCs are cultured with medium that support growth of undifferentiated cells, while detection of 2-AAA in culture medium can be a sensitive early biomarker for differentiation. Once the cells undergo differentiation, PSCs in culture may not be useable for further differentiation procedures because differentiation is an irreversible process. Therefore, monitoring and analytic technologies¹⁵ are important elements to assure the manufacturing quality of cell products and a fundamental pillar for the introduction of quality by design (QbD)-based cell manufacturing.

(1) Reagents

Vitronectin, truncated recombinant human (A14700; Thermo Fisher Scientific)

CTS Essential Medium™ (A2656101; Thermo Fisher Scientific)

StemFit® AK03N (Ajinomoto)

StemFit® Basic03 (Ajinomoto)

ReproFF2 (RCHEMD006; ReproCELL)

Human bFGF recombinant (SP-FGF2-G-001MG for StemFit® Basic03; Ajinomoto and RCHEOT002 or RCHEOT003 for ReproFF2; Reprocell)

ROCK inhibitor (18190-54; Nacalai Tesque)

DPBS (-) (GMP grade; supplier unspecified)

CT TrypL Select Enzyme™ (A1285901; Gibco)

TrypLE Select Enzyme (1x) without phenol red (12563-011; Gibco)

Fixation/Permeabilization solution (554722; BD Bioscience)

Perm/Wash^TM buffer (554723; BD Biosciences)

Rabbit pAb anti-CHD7 (NBP2-41133; Novus)

Rabbit Iso IgG (ab37415; Abcam)

Human serum albumin (25% I.V. 12.5 g/50 mL, Benesis, Japan Blood Products Organization)

Fetal bovine serum (12303; JRH Biosciences)

Total RNA Isolation Kit (supplier unspecified)

TaqMan Gene Expression Assays (assay ID: Hs00215010_m1; Thermo Fisher Scientific)

Droplet Digital PCR (ddPCR) Probe Supermix (186-3024; Bio-Rad)

2-isopropylmalic acid (333115, 100 mg; Sigma Aldrich)

Ultrapure water (39253-1L-R; Sigma Aldrich)

LC-MS grade acetonitrile (34967, 2.5 L; Sigma Aldrich)

MitoProbe™ JC-1 Assay Kit (M34152; Thermo Fisher Scientific)

MitoSOX Red Mitochondrial Superoxide Indicator (M36008; Thermo Fisher Scientific)

CellROX Green Reagent (C10444; Thermo Fisher Scientific)

Systox® Red Dead Cell Stain Solution (S34859; Thermo Fisher Scientific).

(2) Consumables

Sterile pipettes (variable size; supplier unspecified)

Sterile tips for micropipette (variable size; supplier unspecified)

Sterile centrifuge tubes (variable size; supplier unspecified)

Cell culture flasks (surface treated for cell culture, variable size; supplier unspecified)

Low-attachment 6-well plates (3471; Corning)

Cell scraper (9000-220; Iwaki)

Microtubes (1.5 mL; supplier unspecified)

Electric pipette (supplier unspecified)

Micropipette (supplier unspecified)

Biosafety cabinet (class II type B2 or upper model; supplier unspecified)

CO₂ incubator (supplier unspecified)

Phase contrast inverted microscope (supplier unspecified)

Hemacytometer or cell counter (supplier unspecified)

Automated cellular imaging and analysis platform (Cell Insight CX5; Thermo Fisher Scientific)

Droplet digital PCR (QX100; Bio-Rad Laboratories)

Thermocycler (GeneAmp 9700; Applied Biosystems)

Triple quadrupole LC-MS/MS (LCMS-8050; Shimazu Corp.)

FACS Aria II cell sorter (BD Biosciences)

Fluorescent microscope (IX71; Olympus or BZ-X810; Keyence)

1. Cell harvesting and passaging

(1) Preparation of culture media

Glycolytic pathway:

Prepare Essential 8, StemFit® AK03N, or *StemFit® Basic03 as described by the manufacturer (*add bFGF to 80 ng/mL for StemFit® Basic03)

OXPHOS pathway:

Prepare ReproFF2 as described by the manufacturer (add bFGF to 5 ng/mL).

(2) Notes

Some hPSC lines may require an adaptation step for the specific culture media and/or feeder-free conditions using the same procedure as described below, until stabilized. The volume of media and reagents is dependent on the size of culture vessels.

(3) The experimental procedure

1. Prepare hPSCs cultured to be sub-confluent or below at passaging timing.

2. Coat the surface of culture vessels with rhVTN-N (to be diluted to 0.5 μg/cm² in PBS) for 1 h at 37℃ or overnight at 4℃.

3. Warm up aliquots of culture media, TrypLE Select and DPBS (-) in a water bath at 37℃.

4. Remove culture medium by aspiration and wash cells once with DPBS (-).

5. Add TrypLE Select to cover culture surface.

6. Remove excess amount of TrypLE Select (keep surface wet) and incubate at 37℃ in a CO₂ incubator until hPSC colonies are detached from the culture surface.

7. Add culture media and dissociate cells by gentle pipetting to a single-cell suspension.

8. Wash the cells once by gentle spinning down and resuspending in a standard manner, then resuspend the cells in fresh media by gentle pipetting to a single-cell suspension.

9. Count the number of cells on a hemacytometer or cell counter using standard procedures.

10. Seed the cells into pre-coated culture vessels (e.g., at 1.0 × 10⁵ cells/well with Essential 8 medium, 0.8 × 10⁵ cells/well with StemFit medium, or 3.0 × 10⁵ cells/well with ReproFF2 medium) in 6-well plates at 4 mL/well; add ROCK inhibitor only to seeding media to a final concentration of 10 μM.

11. Start culturing cells at 37℃ in an atmosphere containing 5% CO₂; do not move culture vessels until cells are attached to the culture surface.

12. Change culture medium every day; do not add ROCK inhibitor to culture media in this step.

13. 3 days after seeding, harvest, wash, and resuspend the cells for passaging or analysis, as described above.

2. Embryoid body (EB) formation assays

(1) The experimental procedures:

1. Prepare pre-cultured hPSCs with a medium supporting the glycolytic or OXPHOS pathway.

2. The required number of hPSCs for EB formation assay is 1.8 × 10⁶ cells/well in 6-well plates for each condition.

3. Remove the culture media by aspiration and rinse the cells with DPBS (-).

4. Replace DPBS (-) with 2 mL of the same type of culture media containing ROCK inhibitor at 10 μM.

5. Harvest the cells with a sterile cell scraper.

6. Break cellular clumps into smaller pieces with a 5-mL pipet by gentle pipetting 2 times, then transfer into 1 well of a new 6-well plate with a low-attachment surface.

7. Rinse the original wells with fresh medium (the same medium as step 4 above) and combine with cell suspension from step 6.

8. Transfer the well contents into a 15-mL centrifuge tube the next day, spin 2 min at 190 ×g.

9. Carefully aspirate supernatant without aspirating the cellular mass.

10. Add 4 mL of fresh culture medium without ROCK inhibitor and return the cellular suspension into the original well.

11. Replace one-half of the volume with fresh culture medium every 3 days until day 14 of incubation.

12. Measure size and number of EBs derived from hPSCs on days 2 and 14 with a CX5 high-content screening platform (Thermo Fisher Scientific) to measure differentiation potential.

13. [optional] the gene expression profile can be assessed using RT-qPCR with a TaqMan Scorecard Panel (A15870; Thermo Fisher Scientific), and the level of gene expression can be determined using a RT-qPCR device (QuantStudio 12K Flex, Thermo Fisher Scientific).

3. FACS analysis of CHD7 expression

(1) Preparation of reagents

Primary antibody: rabbit pAb anti-CHD7 (1 mg/mL, NBP2-41133; Novus)

Isotype control: rabbit iso-IgG (5 mg/mL, dilute 5 times with DPBS (-), ab37415; Abcam).

Secondary antibody: Alexa Fluor 488 goat anti-rabbit IgG [H+L] (2 mg/mL, A11034; Thermo Fisher Scientific)

Fixation/Permeabilization Solution (554722; BD Biosciences)

Perm/Wash buffer™ (554723, dilute 10 times with DW and use as 1×BD Perm/Wash Buffer; BD Biosciences)

Dilute FBS or HSA with DPBS (-) to 2%.

(2) The experimental procedure

Preparation of cells for FACS analysis:

1. Prepare hPSCs cultured at 80% confluency (1×10⁶ cells in each well of a 6-well plate are required for anti-CHD7 and isotype antibodies).

2. Remove culture medium by aspiration and wash the cells with 2 mL of DPBS (-).

3. Remove DPBS (-) and add 300 μL of TrypLE Select to cover the entire culture surface.

4. Incubate the cells at 37℃ for 3 min in a CO₂ incubator.

5. Check the cellular status for loose colonies, but still attaching on the culture surface.

6. Add 2 mL of PBS to each well and transfer all the contents to a 14-mL centrifuge.

7. Spin down cells at 440 ×g for 5 min at room temperature.

8. Aspirate the supernatant and add Fixation/Permeabilization solution (100 μL per 1×10⁶ cells in a 1.5-mL microtube).

9. Incubate the cells on ice for 30 min.

10. Aadd 1 mL of 1×BD Perm/Wash^TM buffer and spin down the cells at 835 ×g for 2 min, then remove the supernatant by aspiration and repeat the same procedure (2 washes in total).

Immunostaining:

1. Resuspend the cells in 1×BD Perm/WashTM buffer at 1×10⁶ cells/50 μL.

2. Add primary antibody or isotype control at 2.5 μL per 1×10⁶ cells and mix gently.

3. Incubate the cells at room temperature in a dark place for 30 min.

4. Add 1 mL of 1×BD Perm/WashTM buffer and spin down the cells at 835 ×g for 2 min, then remove the supernatant by aspiration and repeat the same procedure (2 washes in total).

5. Resuspend the cells in 50 μL of 1×BD Perm/WashTM buffer.

6. Add secondary antibody at 1.0 μL per 1×10⁶cells and mix gently.

7. Incubate the cells at room temperature in a dark place for 30 min.

8. Add 1 mL of 1×BD Perm/WashTM buffer and spin down the cells at 835 ×g for 2 min, then remove the supernatant by aspiration and repeat the same procedure (2 washes in total).

9. Resuspend the cells in 500 μL of 2% HAS or FBS.

10. Analyze the cells by FACS. Alexa Fluor® 488 fluorochrome emission (519 nm when excited at 488 nm) is collected at the same instrument settings as fluorescein isothiocyanate (FITC).

4. Droplet digital PCR for CHD7-copy number measurement

(1) The experimental procedure

1. Extract total RNA and synthesize cDNA using standard procedures (Qiagen: RNeasy® Mini Handbook and QuantiTect® Reverse Transcription).

2. 5 ng of RNA from each cellular sample were amplified using a TaqMan Gene Expression Assay (assay ID: Hs00215010_m1; Thermo Fisher Scientific) in an Applied Biosystems GeneAmp 9700 Thermocycler.

3. The PCR mixture was loaded into a Bio-Rad QX-100 emulsification device, and droplets were formed following the manufacturer’s instructions.

4. Each 20 μL reaction for droplet formation contained 10 μL of Droplet Digital PCR (ddPCR) Probe Supermix, 1000 nM primers, 250 nM probe, and template cDNA.

5. PCR was carried out using the following cycling conditions (10 min at 95℃, 40 cycles of 30 s denaturation at 94℃ and 60 s of annealing/extension at 53℃, and a final incubation for 10 min at 98℃).

6. After cycling, raw fluorescence data from each well were measured and exported to the manufacturer’s software (Bio-Rad QuantaSoft, ver. 1.2) for analysis.

5. LC/MS analysis of cellular metabolites in culture media

(1) Notes

Conduct this experiment using chemically-defined culture media, such as Essential 8 or TeSR-E8 medium, to eliminate high analytical background noise generated by medium components, such as bovine serum albumin (BSA) and human serum albumin (HSA).

(2) Sample pre-processing

1. Centrifuge the culture media to remove cellular debris and a 100-μL aliquot of each was transferred into a new tube.

2. Add 20 μL of an internal standard solution (0.5 mM 2-isopropylmalic acid dissolved in ultrapure water and 200 μL of LC-MS grade acetonitrile to each tube).

3. Thoroughly agitated the solution and centrifuged at 20,400 ×g for 15 min at room temperature.

4. Transfer 100 μL of the supernatant into a new tube, dilute the supernatant with 900 μL of ultrapure water, and thoroughly agitate for LC-MS/MS analysis.

(3) LC-MS/MS analysis

1. Analyze pretreated samples in multiple reaction monitoring (MRM) mode using triple quadrupole LC-MS/MS (LCMS-8050) and the Cell Culture Profiling Method Package (Shimadzu Corp.), which enabled us to simultaneously analyze 95 compounds, including basal medium components and secretion of metabolites in 17 min.

2. For normalization of the measured area ratio, collect the culture medium every 24 h, refed the cells with fresh medium, and draw cellular growth curve by plotting the cell number counted at each medium collection.

3. Calculate the amounts of secreted metabolites from a single cell in 1 h by dividing the area ratio or μM measured by LC-MS/MS with the area defined by the cell number curve of the past 24 h (area ratio/cell/h) or (nmol in a 2-mL culture volume/cell/hour).

6. JC-1 mitochondrial membrane potential flow cytometry

(1) The experimental procedure

1. Harvest hPSCs and dissociate to a single cell preparation using TrypLE Select.

2. Resuspended the cells at 1 × 10⁶ cells in 1 mL of warm medium

3. Only reference cells are to be incubated after the addition of 1 μL of 50 mM carbonyl cyanide 3-chlorophenylhydrazone at 37℃ for 5 min.

4. Incubate sample and reference cells after addition of 10 μL of 200 μM JC-1 at 37℃ for 15 min.

5. Wash the cells twice with warm DPBS (-) after incubation.

6. Analyze the cells using a FACS Aria II cell sorter (BD Bioscience) and FlowJo software (FlowJo LLC); use 488 nm excitation with 530/30 nm and 585/42 nm bandpass emission filters.

7. Measurement of ROS levels in live cells

(1) The experimental procedure

1. Stain cultured cells (adherent or harvested) with MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific) and CellROX Green Reagent (Thermo Fisher Scientific), as described by the manufacturer.

2. In the case of flow cytometry, add SYTOX® Red Dead Cell stain solution at the last 15 min of incubation

3. The standard excitation/emission wavelengths are 510/580 nm for MitoSOX Red, 508/525 nm for CellROX Green, and 640/658 nm for SYTOX® Red.

4. Capture microscopic images using fluorescent microscope for live-cell imaging; conduct flow cytometry analysis and sorting with a FACS ARIA II (BD Biosciences).

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3 Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science (New York, N.Y.) 318, 1917-1920, doi:10.1126/science.1151526 (2007).

4 Kim, J. Y., Nam, Y., Rim, Y. A. & Ju, J. H. Review of the Current Trends in Clinical Trials Involving Induced Pluripotent Stem Cells. Stem Cell Rev Rep 18, 142-154, doi:10.1007/s12015-021-10262-3 (2022).

5 Osafune, K. et al. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat Biotechnol 26, 313-315, doi:10.1038/nbt1383 (2008).

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9 Yamamoto, T. et al. Correlation Between Genetic Abnormalities in Induced Pluripotent Stem Cell-Derivatives and Abnormal Tissue Formation in Tumorigenicity Tests. Stem Cells Transl Med 11, 527-538, doi:10.1093/stcltm/szac014 (2022).

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11 Yamamoto, T. et al. Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7. Sci Rep 8, 241, doi:10.1038/s41598-017-18439-y (2018).

12 Yamamoto, T., Arita, M., Kuroda, H., Suzuki, T. & Kawamata, S. Improving the differentiation potential of pluripotent stem cells by optimizing culture conditions. Sci Rep 12, 14147, doi:10.1038/s41598-022-18400-8 (2022).

13 Takenaka, C. et al. Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes. PloS one 10, e0129855, doi:10.1371/journal.pone.0129855 (2015).

14 Yamamoto, T. et al. Kynurenine signaling through the aryl hydrocarbon receptor maintains the undifferentiated state of human embryonic stem cells. Sci Signal 12, doi:10.1126/scisignal.aaw3306 (2019).

15 CellQualia - INTELLIGENT CELL PROCESSING SYSTEM https://www.cellqualia.com/eng/.

We thank H. Itaya and S. Wakimoto of Ajinomoto Co., Inc. for medium supply and critical discussion, and H. Kuroda and T. Suzuki of Shimadzu Co. for LC-MA/MS measurement and critical discussion.

Contribution: T.Y. conducted the experiment and prepared the manuscript, M. H. wrote the manuscript, and S. K. supervised all experiments and manuscript preparation.

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Standardization of human-induced pluripotent stem cells by optimizing culture conditions and monitoring key parameters

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