Standardized quality control workflow to evaluate the reproducibility and differentiation potential of human iPSCs into neurons

Induced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a multistep workflow to assess our newly generated iPSCs for variations and reproducibility relative to each other and iPSCs obtained from external sources. Our benchmarks for evaluating iPSCs include examining iPSC morphology and proliferation in two different media conditions and evaluating their ability to differentiate into each of the three germ layers, with a particular focus on neurons. Genomic integrity in the human iPSCs was analyzed by G-band karyotyping and a qPCR-based test for the detection of hotspot mutations test. Cell-line identity was authenticated by Short Tandem Repeat (STR) analysis. Using standardized dual SMAD inhibition methods, all iPSC lines gave rise to neural progenitors that could subsequently be differentiated into cortical neurons. Neural differentiation was analyzed qualitatively by immunocytochemistry and quantitatively by qPCR for progenitor, neuronal, cortical, and glial markers. Taken together, we present a multistep quality control workflow to evaluate variability and reproducibility across and between iPSCs.


Introduction
Human pluripotent stem cells can give rise to any cell type when exposed to the appropriate developmental cues, holding enormous potential for tissue engineering, regenerative medicine and disease modeling. The growing number of iPSC lines and NIH-registered human embryonic stem cell (ESC) lines ensures that patient-derived pluripotent stem cells are now readily available to researchers, helping to accelerate our understanding of biology and disease and the development of therapies across disease areas [1]. These developments underscore the need for iPSC quality standards that are sufficiently stringent to ensure that findings can be compared and results reproduced across laboratories [2,3].
For instance, it is critical that the growth parameters of a given iPSC cell-line are optimized.
Since the advent of iPSC technology, culture conditions have become more standardized, helping to increase reproducibility between and within cell-lines. Several media formulations have been developed to grow and maintain iPSCs [4,5]. Thus, depending on the media used, iPSC growth can vary, making it imperative that the optimal growth media and maintenance conditions be defined before working with any new cell-line and to reduce variation between lines [5][6][7][8]. Moreover, numerous studies on pluripotent ESCs and iPSCs have identified genomic integrity as a critical issue when monitoring the quality of cells for use in research or clinical applications. Common alternations that arise in the genome during iPSC reprogramming, from culture conditions [9] or off-target CRISPR/Cas9 genome editing [10,11] can include, but are not limited to, (1) chromosomal/karyotypic abnormalities [12][13][14] (2) copy number variations (CNVs), (3) small genomic insertions and deletions, or (4) single nucleotide variants [15]. When present, these genomic abnormalities often alter the biological properties of hiPSC-derived models. Thus, before a given iPSC is used, it should be tested for alterations at the genomic level [3,16,17].
Nonetheless, each benchmark for evaluating iPSCs has its own strengths and limitations in terms of sensitivity, costs, time and effort. Considering these points, we have developed a rigorous quality control workflow to evaluate newly generated iPSC lines. In this paper, we present a workflow combining these quality control (QC) assays that we used to test for variability across ten newly generated hiPSC lines derived from healthy individuals, relative to existing commercial lines. With these cell-lines, we established a workflow to monitor hiPSC morphology and proliferation in two different media. As well, the iPSCs were authenticated through Short Tandem Repeat (STR) profiling, to verify that each iPSC line generated matched the parental cell from which it was reprogrammed. Genomic integrity in the hiPSCs was analyzed by G-band karyotyping and qPCR-based profiling for genomic hotspot regions that are commonly altered during reprogramming [18][19][20]. We also evaluated the pluripotency of our lines by examining their ability to form embryoid bodies (EBs), to differentiate into each of the three germ layers, and their potential to form cortical neurons. We focused on cortical neurons, as methods to generate these neurons from iPSCs are well established, and represent a cell type broadly studied across disease areas of the brain, from Alzheimer's disease to neurodevelopmental disorders [21,22]. While all iPSC lines were capable of generating cortical neurons under standard differentiation protocols [23], line-to-line variability in the number of neurons formed, and their overall morphology was observed, highlighting the importance of deep phenotyping of cell lines before they are used in a given downstream application. Taken together, we developed a multistep, QC workflow (Figure 1) to validate our newly generated iPSCs lines and their ability to reproducibly differentiate into human cortical neurons for downstream applications. Ensuring iPSC colonies maintain an undifferentiated morphology, determining the growth rate of each line, and assessing the pluripotency and differentiation potential of each line are essential quality control measures, to reduce variability across different iPSCs.

Culture conditions for iPSCs
iPSCs were cultured and expanded on plates coated with Matrigel (Corning, 354277) in either mTeSR1 or E8 (ThermoFisher Scientific, A1517001) media. Cells were maintained at 37 o C with 5% CO2 with daily media changes and split when cells reached 70-80% confluency (within 5-7 days of seeding). Any iPSC colonies with irregular borders, spontaneous differentiation or transparent centers were manually removed prior to splitting. Cells were passaged by incubation in Gentle Cell Dissociation media (Stemcell Technologies, 07174) for 4 minutes at 37 o C to obtain single cells or RT for 6 minutes to obtain small aggregates of colonies. The following cell densities were used: 2×10 4 cells/well in 24 well plates for immunocytochemistry; 2×10 4 /60 mm dish for daily morphology imaging; 2×10 5 /well in 6 well plates for EB formation.

Crystal violet assay
For this assay, 4000 cells/well were plated in Matrigel-coated 96-well plates with mTeSR1 or E8 media. After 2, 4 or 6 days in culture, plates were rinsed with PBS to remove nonattached cells and fixed with 3.7% PFA for 5 minutes, before staining with 0.05% crystal violet (CV, Sigma,46364) diluted in water for 30 minutes. The CV dye was thoroughly washed away using distilled water, and the plates dried at RT. Once dried, the plates were imaged and quantification performed [25] by adding 100 µl of methanol (Fisher Chemical, 32435K7) to the wells to solubilize the CV, followed by measurement of the O.D. at 540 nm (OD540) with an EnSpire Multimode Plate Reader (Perkin Elmer).

RNA isolation, cDNA synthesis, and qPCR analysis
RNA was purified with a NucleoSpin RNA kit (Takara) according to the manufacturer's instructions. cDNA was generated using the iScript Reverse Transcription Supermix (BioRad).
Quantitative real-time PCR was performed on the QuantStudio 5 Real-Time PCR System (Applied Biosystems) using the primers listed in Table S1. Raw data was processed using a custom Python script available at https://github.com/neuroeddu/Auto-qPCR. The cycle threshold (CT) values for technical triplicates were tested for outliers. Relative gene expression was calculated by using the Comparative CT Method (ΔΔCT method), where the endogenous controls were GADPH or ACTB expression. The reference sample varied by experiment and is indicated in each plot.

Short Tandem Repeat (STR) analysis
All newly generated iPSCs were authenticated through STR analysis with the GenePrint® 10 System (Promega, B9510) at The Centre for Applied Genomics, the Hospital for Sick Children, Toronto. Briefly, genomic DNA from the iPSCs or the source material for the iPSCs, which in this case was PBMCs, was extracted with a Genomic DNA Mini Kit (Blood/Cultured Cell) (Geneaid, GB100). Ten ng of genomic DNA was mixed with GenePrint® 10 primer pair mix to permit co-amplification and detection of ten human loci, including all ASN-0002-2011 loci (TH01, TPOX, vWA, CSF1PO, D16S539, D7S820, D13S317, D5S818) plus Amelogenin for gender identification and one mouse locus D21S11. These loci collectively provide a genetic profile with a random match probability of 1 in 2.92×10 9 .

Karyotyping and genomic abnormalities analysis
Genomic DNA was extracted with the Genomic DNA Mini Kit. Genomic integrity was detected with the hPSC Genetic Analysis Kit (Stemcell, 07550) according to the manufacturer's instructions. Briefly, 5 ng of genomic DNA was mixed with a ROX reference dye and double-quenched probes tagged with 5-FAM. The probes represented eight common karyotypic abnormalities that have been reported to arise in hiPSC: chr 1q, chr 8q, chr 10p, chr 12p, chr 17q, chr 18q, chr 20q or chr Xp. Sample-probe mixes were analyzed on a QuantStudio 5 Real-Time PCR System (ThermoFisher Scientific). Copy numbers were analyzed using the ΔΔCt method. The results were normalized to the copy number of a control region in chr 4p [20]. For G-band karyotyping, iPSCs were cultured for 72h until they attained 50-60% confluency, then were shipped live to the Wicell Cytogenetics Core (instructions provided by WiCell).

Three Germ Layer Differentiation Test
To form EBs, 2 wells of a 6 well plate containing 80-90% confluent iPSCs were dissociated into small clumps and cultured on low-attachment tissue plates in their preferred iPSC maintenance media (based on CV assays in 2.4.). On day 7, EBs were transferred to Matrigel- To differentiate iPSCs into each of the three germ layers, cells were passaged and dissociated as described above for EB generation into single cells and cultured on Matrigelcoated plates with the STEMdiff Trilineage differentiation kit (used according to the manufacturer's instructions, Stemcell Technologies, 05230). Cells were harvested at the indicated day for gene expression analysis by qPCR.

Immunocytochemistry analysis
Cells were fixed in 4% PFA/PBS at RT for 20 minutes, permeabilized with 0.2% Triton X-100/PBS for 10 min at RT, then blocked in 5% donkey serum, 1% BSA and 0.05% Triton X-100/ PBS for 2h. Cells were incubated with primary antibodies in blocking buffer overnight at 4 °C. Secondary antibodies were applied for 2h at RT, followed by Hoechst 33342 nucleic acid counterstain for 5 minutes. Immunocytochemistry images were acquired using the automated Evos FL-Auto2 imaging system (ThermoFisher Scientific). Antibodies used for staining are listed in Table S2. Images were quantified using custom ImageJ macros. The analysis scripts are available at https://github.com/neuroeddu/CellQ. The thresholds were determined visually by comparing five randomly sampled images.

Cortical neuron differentiation
Differentiation into cortical neurons was based on a protocol for EB formation combined with dual inhibition of SMAD [23,26] with modifications. Briefly, each iPSC line was cultured in its preferred media for 5-7 days, then dissociated into single cells to form EBs. The EBs were grown in a low-attachment plate for one week in DMEM/F12 supplemented with N2 and B27, in the presence of 10 μM SB431542, and 2µM DMH1. On day 7, EBs were transferred to polyornithine-and laminin-coated plates to form rosettes in the same media. On day 14, rosettes were selected semi-manually and cultured as a monolayer on polyornithine and laminin-coated plates to generate neural progenitor cells (NPCs) in DMEM/F12 supplemented with N2 and B27. NPCs were passaged at a 1:3 dilution every 5-7 days. Immunocytochemistry and qPCR analysis of NPC were conducted at day 25. NPCs were next cultured in neurobasal medium, supplemented with N2 and B27, in the presence of 1 μg/ml laminin, 500 μM db-cAMP, 20 ng/ml BDNF, 20 ng/ml GDNF, 200 μM ascorbic acid, 100 nM Compound A and 1 ng/ml TGF-β for differentiation into neurons. Immunocytochemistry and qPCR analysis of cortical neurons were conducted at day 56.

Data visualization and statistical analysis
All data visualization plots were created in R using the ggplot2 graphical package. For

Validation of hiPSC culture conditions
To work with any iPSC line, it is critical to first establish the optimal growth conditions for that cell-line. As a first step in our workflow, and as part of the initial tests for our newly reprogrammed iPSCs, we grew all our newly generated iPSCs cell-lines side by side in two distinct maintenance media: mTeSR1 or E8. Commercial iPSC lines (NCRM1-NIH and KYOU-DXR0109B-ATCC) were also grown in both media for direct comparison with our "in house" control lines (see cell line profiles in Table 1). Cells were seeded at an identical confluency and cultured in Matrigel-coated plates in either mTeSR1 or E8 media. We first examined cells 24h after passaging for differences in their overall attachment, spontaneous differentiation and morphology as they grew and expanded. All iPSC lines attached onto Matrigel-coated plates and demonstrated similar morphology when maintained in either media condition. The condensed, round, diffuse and irregularly shape associated with iPSC colonies was observed across all cell lines. The colonies were smooth-edged, with tightly packed cells observed by phase contrast imaging ( Figures. 2A and 3A). Rare incidences of spontaneous differentiation, in which the cells lost their pluripotency, could be observed with both media conditions and across the different cell-lines.
Although morphology is one indicator of iPSC quality, growth properties can vary from line to line, which can be influenced by the media. To detect differences in growth rates and adherence of cells over time, cells were fixed and stained with crystal violet (CV) (Figures. 2B and S1) and demonstrate that the growth rate of all cell-lines in each media condition was comparable at day 2. By day 4, three lines were observed to be growing at a faster rate in E8 (AJD002-3, 3450 and TD10; Figures. 2C and S1) and exhibited reduced growth and proliferation when maintained in mTeSR1. By day 6, the difference in growth rates was further pronounced for these lines, with 3448 and TD22 also demonstrating a preference for E8. Of note, the proliferation rate for AJG001-C4 was the slowest across the lines, growing at a comparable rate in both media ( Figures. 2C and S1). For our panel of iPSCs, variations in the growth rate between certain iPSCs was observed depending on the media conditions. Thus, in the rest of our assays, the cells were grown in their preferred media ( Table 1).
Taken together, daily morphological observations coupled with a CV assay, enable a rapid and economical assessment of the growth conditions for a given cell-line, ensuring that each iPSC is cultured in its optimal media for growth and expansion.

Characterization of iPSC pluripotency
Next, we examined the pluripotency of each iPSC line in its preferred growth media (mTeSR1 or E8), as determined by the CV assays ( Figure.  Notably, we found that over 90% of cells were OCT3/4 or SSEA-4 positive, demonstrating that the cells were maintained in a state of pluripotency with rare spontaneous differentiation ( Figure. 3B). iPSCs were not only morphologically similar each other and to ESCs but were also similar at a transcriptional level for a number of pluripotency markers that can be commonly observed (OCT3/4, SOX2, NANOG) [27][28][29][30][31]. To examine the transcriptional profile of our lines, the embryonic stem cell, H9 was used as a reference line to normalize the expression of pluripotency genes relative to each of the control iPSC lines [32]. The expression of the Yamanaka factors (OCT3/4, SOX2, KlF4 and c-MYC) and two other widely tested pluripotent markers, NANOG and ZFP42 (Rex1) [33] were analyzed by qPCR ( Figure. 3C). We found that all iPSC lines expressed each of the pluripotency genes, and expression levels for each gene was comparable to levels in the H9 ESC cell-line.
However, variations in expression of the genes was observed between the lines. We found that NCRM1 and KYOU-DXR0109B, two commercial control lines with high passage numbers, expressed relatively high levels of NANOG, SOX2 and ZFP42 when compared to H9, suggesting that prolonged periods of proliferation and self-renewal may stabilize and perpetuate the genome of the hiPSC in a pluripotent state. Given our iPSCs are at an earlier passage number, these levels were lower, consistent with previous studies showing that hESC-specific genes [34] are expressed at lower levels in early passage iPSCs compared to ESCs and late passage iPSC [35].

STR and genomic abnormality testing of hiPSCs
The risk of cell misidentification and cross-contamination has plagued cell research [36,37].
In addition, hiPSCs which are generated and grown on a layer of mouse embryonic fibroblasts (MEF) feeder cells are often at risk of cross-contamination with non-human rodent somatic cells [38]. For this reason, we conducted STR analysis for iPSC authentication. Our analysis demonstrated that the STR profile for each iPSC tested matched the parental cell-line from which it was reprogrammed, and no rodent contamination was detected with any of our iPSCs.
As both AJD002-3 and TD03 were generated from the same donor, they displayed identical STR profiles relative to each other, and to somatic cells obtained from this donor (PBMC sample #3059) ( Table 2).
Numerous studies have demonstrated that ESCs and iPSCs accumulate genomic alterations and mutations though reprogramming processes and long-term culture. At the genome level these changes can include copy number variations, trisomy's, small genomic insertions and deletions, and single nucleotide variants. To assess chromosomal integrity of the iPSC lines, G-band karyotyping was performed. Our analysis showed that the majority of iPSCs tested had a normal 46, XY or 46, XX karyotype ( Figures. 4A and S2). However, we did detect a chromosomal anomaly in the TD10 line, which contained a translocation between the long (q) arm of chromosome X and the short (p) arm of chromosome 2. These abnormalities were confirmed to be a direct result of reprogramming, as follow-up G-band analysis of the parental PBMCs showed a normal karyotype (data not shown). This confirms that the chromosomal rearrangement in TD10 likely occurred during the reprogramming process and disqualifies this line from further use.
Although karyotyping by G-banding reveals both numerical and structural aberrations within chromosomes, the limited resolution of this method means we can only detect chromosomal aberrations greater than 5 Mb [14,39,40]. To test for commonly occurring genomic alterations, we used a qPCR-based genetic analysis kit to detect minimal critical hotspot regions within the genome that are frequently mutated during the reprogramming process and extended cell passaging, often conferring selective growth advantages to the cells [41,42]. When testing our newly generated iPSCs, we did not detect any increase or decrease in copy number outside the confidence interval (1.8 to 2.2) indicating there are no abnormalities in any of the eight common hotspot zones tested on chr 1q, chr 8q, chr 10p, chr 12p, chr 17q, chr 18q, chr 20q and chr Xp with our "in house" iPSCs, which cover the majority of the reported abnormalities ( Figure. 4B) [21,[43][44][45][46], except for a moderate increase in the copy number for chr 20q in our TD03 line ( Figure. 4B, indicated with #). However, with both the commercial lines, NCRM1 and KYOU-DXR0109, an amplification in copy number on chromosome 20q was detected (Figure. 4B, indicated with arrows), suggesting that this abnormality expanded with extensive cell passage. Thus, it is imperative that when working with any iPSC line, they are profiled for the presence of genomic alterations, which might otherwise affect its growth and differentiation.

Differentiation of hiPSCs into three germ layers
One of the hallmark features of iPSCs is their ability to differentiate into nearly any cell type of the three germ layers when provided with the appropriate developmental cues. To characterize the functional pluripotency of our newly generated iPSCs relative to the commercial lines, we tested their ability to form EBs, in which cells spontaneously differentiate into each of the three embryonic germ layers. After one week, all iPSCs successfully formed EBs and no discernible differences in the relative size and total number of EBs formed was detected between lines. Following the formation of EBs in defined media, they were plated onto Matrigel-coated dishes in the presence of serum-containing media and cultured for 2 weeks. All iPSCs tested could differentiate into each of the three germ layers, as shown by positive immunostaining for the ectoderm marker PAX6, the mesoderm marker SMA and the endoderm marker Vimentin (Figure. 5A).
In parallel to our image analysis, we also took advantage of a qPCR based assay to permit a faster, more quantitative assessment of functional pluripotency. Through this approach, we were able to quantify the in vitro differentiation potential of our hiPSCs by measuring the relative expression of key genes that represent each of the three specific lineages. We used the H9 ESC line as our control to compare the expression of three germ layer genes in the iPSC lines. As shown in Figure.  In contrast, TD10 expressed all the lineage markers at relatively high levels, indicating that the presence of the karyotypic abnormality had no effect on its ability to differentiate and form each of the three germ layers. ICC staining and qPCR findings confirmed that all iPSCs were capable of differentiation into each of the three germ layers. However, based on the expression analysis, the abilities of iPSC to generate different cell types was variable, highlighting the need to choose iPSC lines carefully based on the cell types to be generated for downstream applications.

Differentiation of iPSCs into cortical neurons
Following our trilineage analysis, we narrowed our focus from all germ layers down to one specific cell type from the ectodermal lineage, cortical neurons. To generate cortical neurons, we used a previously published dual SMAD inhibition protocols with modifications [23,26].   Pluripotency (POU1F5 and NANOG), mesodermal (MIXL1) and endodermal (AFP) markers were expressed at low levels, which was to be expected given the cell type generated was of an ectodermal lineage ( Figures. S4A and S4B). The expression of the NPC marker Nestin was further confirmed by qPCR ( Figure. S4C). NPCs also expressed dorsal forebrain progenitor (SLC1A3 and PAX6), and ventral forebrain progenitor (ASCL1) markers ( Figure. S4C), which confirms that the iPSCs tested can differentiate into NPCs [23,47].
NPCs generated from each line were subsequently differentiated into cortical neurons that we analyzed through a combination of qPCR and ICC analysis (Fig. 6A). qPCR results confirmed high expression levels of the neuronal markers MAP2, NCAM1 and Tuj (TUBB3) in neurons at day 56 differentiation (Fig.6B). Furthermore, upper layer (SATB2) or lower layer (FOXP1) cerebral cortex markers were detected in neurons (Fig. 6C). However, the expression level of these markers exhibited line to line variation. Intriguingly, we compared expression levels in the neurons derived from iPSCs lines maintained in mTeSR1 to neurons from iPSCs maintained in E8 and found that levels of these markers was significantly higher for neurons derived from iPSCs maintained in mTeSR1 (p= 0.0349 MAP2; p=0.017 NCAM1; p= 0.018 TUBB3; p = 0.008 FOXP1 and p = 0.010 SATB2) (Fig. S5). The higher expression levels of these markers implies that the iPSC maintenance media may influence the differentiation potential of iPSCs, and in this case specifically, into cortical neurons, meriting further investigation. Based on the quantification of the immunostained neurons, approximately 80-90% of cells expressed MAP2 and 60-80% of cells were Tuj1-positive neurons, varying from line to line (Fig. 7). In contrast, the expression of the cortical neuron markers Brn2 or Tbr1 was much more variable (5-30%) across cell-lines (Fig. 7). Taken together, the iPSCs tested can generate neurons, although variations in morphology and expression of neuronal or cortical region markers does vary across lines. Thus, such variables may similarly impact iPSC differentiation into other cell types making it imperative that before any studies, an iPSC is comprehensively phenotyped to obtain a baseline on its cellular characteristics.

Discussion
The routine utilization of iPSCs requires a constant supply of pluripotent, well characterized and quality-controlled cell stocks. However, without standardized quality control, experimental reproducibility with iPSCs can be compromised, making findings difficult to interpret [3,16,38].
Here, we established a workflow to monitor the morphology and proliferation of our newly generated iPSCs in two different media (mTeSR1 and E8). In parallel, we evaluated the genomic abnormalities, pluripotency and differentiation potential of our lines. Based on these parameters, we can evaluate whether iPSCs can be used for further applications. However, this is just one iteration of a workflow and the modular nature of the workflow developed means additional quality control tests can be added in further iterations. These can include but are not limited to live cell imaging of iPSCs for growth rate analysis, whole genome sequencing and trilineage analysis through teratoma formation. By focusing on cortical neuron differentiation from the panel of iPSCs, we can also better predict how variable cell-lines are in their ability to generate cortical neurons with the same methods while understanding how the neurons generated might differ from line to line, or from batch to batch within the same line.
Today, commercially available medium are widely used to culture hiPSCs [4][5][6][7][8]. We used CV staining [25] which is directly proportional to cell biomass for our assays which provides a affordable and straightforward method to quantify the proliferation of each cell line at different time points. However, other approaches can be performed in addition to, and to complement CV assays, from live cell imaging of iPSC growth rate, to fixed analysis for growth markers through immunocytometry and flow cytometry if desired. While many of our lines grew in both media at comparable rates, some cell lines had a preference for one of the media tested. Five cell lines grew faster in E8 (Figures. 2B, 2C and S1), while other lines proliferated at a comparable rate in both media tested. The reason is not clear, but it has been demonstrated previously that the reduced composition of E8 (8 factors) can often elicit a faster growth rate [7]. It is also unclear why both the 3450 and TD10 cell-lines grew well in E8, while their growth appeared to stall in mTeSR1. Interestingly, we found that cell lines derived from the same donor (AJC001-5 and AIW002-02, Table 1), albeit different cell types (PBMC vs skin fibroblasts), tended to grow at comparable rates in either media type. Among lines generated from the same donor's PBMCs (AJD002-3 and TD03), the reprogramming methods (Sendai CytoTune vs. episomal) did not appear to affect the preferred media. Thus, before working with any iPSC it is imperative that culture conditions and media are optimized.
Moreover, if a line appears to have a slower growth rate with one media type, it would be recommended to test other media conditions to ensure it's the growth conditions and not the line itself, accounting for the poor growth rate of a given iPSC line.
Expression of pluripotency-associated markers is an important quality criterion for any iPSC line, otherwise the iPSCs cannot be differentiated into a cell type of interest. All iPSC lines in our study expressed the pluripotency markers SSEA-4, OCT3/4, NANOG and TRA-1-60R in defined culture conditions ( Figures. 3A and B). We did find that OCT3/4 expression level was approximately 15 times higher than that of SOX2 (Table S3), consistent with previous studies, showing this OCT3/4 high, SOX2 low stoichiometry is important not only in the early phase of reaching a fully reprogrammed state, but also in the late phase of iPSC maturation and maintaining pluripotency [50][51][52]. Further evidence has also demonstrated that OCT3/4 downregulates the downstream gene expression of NANOG, SPP1/ OPN, SOX2, FBXO15, OTX2, and ZFP42/REX1 [53]. The expression levels of NANOG, ZFP42 and c-MYC in our study were approximately 50 times lower than that of OCT3/4, which is consistent with previous results from single cell RNA-sequencing iPSC dataset [54,55]. Interestingly, NCRM1 and KYOU-DXR0109B, two commercial control lines with high passage number (>30 passages), expressed relatively high levels of NANOG, SOX2 and ZFP42 and reduced levels of OCT3/4, suggesting that extended passaging enhances pluripotent gene expression in an undifferentiated state and increases the efficiency of neuronal conversion [56].
A core feature of hiPSCs is their pluripotency, that is, the ability to differentiate into nearly any cell type of the three germ layers. The previous gold standard method to assess pluripotency of iPSCs was a teratoma assay, in which iPSCs were injected into immunedeficient mice to assess their ability to form teratomas [27,57]. However, this requires the sacrifice of animals, and can be expensive and time consuming, leading to the development of trilineage assays to assess the pluripotency of the cells [58]. One such approach is the In vitro formation of EBs, a commonly used method to assess the differentiation capability of a given iPSC [59,60]. All the iPSCs we tested in our panel had the potential to differentiate into each of the three germ layers, as shown by positive immunostaining for the ectoderm, mesoderm, and endoderm markers PAX6, SMA and Vimentin, respectively ( Figure. 5A), as previously reported [61]. However, while we successfully generated each of the three germ layers, the heterogeneous nature of the EBs resulted in inefficient and often variable differentiation of the three germ layers with each line. To further standardize our protocols, we used a commercial trilineage differentiation kit to perform parallel in vitro directed differentiation experiments for each germ layer. We also took advantage of a qPCR-based assay to enable a faster, more quantitative assessment of functional pluripotency, relative to the image-based approach of the EB trilineage test. Through this approach, we could quantify the in vitro differentiation potential of our iPSCs by measuring the relative expression of key genes that represent each of the three lineages. From this test, all the iPSCs expressed markers for each of the three germ layers, albeit at differing levels, with some lines expressing higher levels of one or more markers compared to each other ( Figure. 5B). Nevertheless, depending on the line itself, some consideration needs to be given as to whether it is the optimal line required to generate a particular cell type of interest.
Numerous studies have demonstrated that both ESCs and iPSCs accumulate genomic abnormalities during long-term culturing, and often is the primary reason why clinical therapies from stem cells were not administered to patients [62]. The presence of genetic variations in iPSCs has raised serious safety concerns for both patient interventions and basic research studies, hampering the advancement of novel iPSC-based therapies. G-banding was widely used for genetic evaluation [14] and upon karyotyping, the majority of iPSCs we tested maintained a normal 46, XY or 46, XX karyotype ( Figures. 4A and S2). However, we were able to detect a genomic anomaly in one of our control lines, TD10 which presented with an abnormal karyotype, that is, a translocation between the long (q) arm of chromosome X and the short (p) arm of chromosome 2. Yet, when compared to other iPSCs, this line appears within the normal range for other parameters, highlighting how important it is to assess each line for genomic abnormalities with multiple tests. One such test we applied in our analysis was a qPCR-based genetic analysis kit to detect minimal critical hotspot regions within the genome that can arise during the reprogramming process or confer selective growth advantages for a given cell-line [41,42]. Using this analysis, which covers the majority of reported abnormalities, we did not detect any abnormalities in any of the hotspot zones with our newly generated iPSCs ( Figure. 4B) [21,[43][44][45][46] . However, with the two commercial lines, NCRM1 and KYOU-DXR0109, an amplification in copy number on chromosome 20q was detected ( Figure. 4B, labeled with an arrow). This region is enriched with genes associated with pluripotency and anti-apoptosis, including DNA methyl-transferase 3B (DNMT3B), inhibitor of DNA binding 1 (ID1), and BCL2-like1 (BCL2L1) [45,63]. We also detected a slight increase in copy number for chr8q in NCRM1, which is a previously reported abnormality acquired during prolonged periods in culture [14], suggesting rigorous quality control is needed with these lines, as it is likely that with increasing cell passage, these abnormalities have expanded. These findings strongly suggest that it is critical to test lines for genomic abnormalities that can arise through reprogramming and prolonged cell passage. Additional quality control tests worth pursued in future iPSC profiling is whole genome sequencing (WGS) [64,65]. With recent advances significantly reducing the cost of WGS to more affordable levels for its widespread use in research labs, it can help in future workflows to detect low frequency variations which could not be identified by conventional methods and adding an extra layer of quality control profiling.
Moving beyond a broad trilineage test, we next tested our lines for their ability to form one specific cell type of interest, that is, cortical neurons. Prior studies have shown that dual SMAD inhibitors synergistically destabilize the activin-and NANOG-mediated pluripotency network [66], suppresses BMP-induced mes-/endodermal fates differentiation [67,68] and promoted neuralization of the primitive ectoderm by BMP inhibition [23]. Taken together, our findings indicate no preference for a specific layer in the generation of cortical neuron by dual SMAD inhibition EB method, yet the expression levels were highly variable across iPSCs. The variability does not come from the differentiation protocol, as all the iPSCs were differentiated under the same conditions. This variability may not simply be a direct result of distinct genetic background differences, since variations in differentiation were also detected between AIW002-02, AJC001-5 and AJG001-C4 which share the same genetic background.
Intriguingly, the iPSCs maintained in mTeSR1, expressed FOXP1 and SATB2 at levels much higher than those maintained in E8, potentially indicating that the conditions we culture the cells might impact their ability to form defined types of neurons, an area which warrants further study.

Conclusions
Reprogramming of somatic cells into iPSCs opens up the possibility of modeling human diseases and developing new therapeutics. Using human iPSCs-derived cells for preclinical and clinical research will require a constant supply of well characterized pluripotent cell-lines.
Thus, in this study, we established a workflow to monitor the growth and morphology of newly generated iPSCs in two different media. We also performed a comprehensive phenotyping of the iPSC lines through growth rate profiling, testing of genome integrity, analysis of pluripotency capacity and tests on each of the iPSCs to form each of the three germ layers, with a particular focus on cortical neurons of the ectodermal lineage. From these studies, we demonstrated that our newly generated iPSC lines share common hallmarks yet can vary in their growth rate or ability to differentiate into other cell types. Given our findings, it is imperative that each new iPSC line be evaluated thoroughly before using it in downstream applications, while ensuring the line can be used to generate the cell types of interest for a given research application. With these parameters in mind, the workflow outlined will help streamline work processes and offers the potential to add new tests as technologies evolve, to ensure researchers employ iPSCs of the highest quality for experimental reproductivity and robustness. Figure S1. HiPSC growth and proliferation profile in mTeSR1 or Essential 8 media; Figure   S2. HiPSCs maintain a normal karyotype; Figure S3. Characterization of hiPSC-derived NPCs; Figure S4. Gene expression profiles in neural progenitor cells; Figure S5. Comparison of gene expression levels in the neurons derived from iPSCs lines maintained in mTeSR1 to neurons from iPSCs maintained in E8; Table S1. Primers used in qPCR experiments; Table   S2. Key resources; Table S3. Quantification of pluripotency gene expression in hiPSCs by qPCR analysis.

Author Contributions
Author contributions were diverse and covered many aspects of the work performed

Institutional Review Board Statement
All subjects gave their informed consent for inclusion before they participated in the study.
The study was conducted in accordance with the Declaration of Helsinki, and the protocol was

Informed Consent Statement
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement
Data are not available for distribution from this study.  Schematic representation of a multistep QC workflow to monitor the morphology and proliferation of newly generated iPSCs, genomic integrity, pluripotency, and an ability to form cells of the three germ layers.