Development and Characterization of the Healthy Control Human iPSC Line, SCTi003-A
Background
In June 2022, the International Society for Stem Cell Research (ISSCR) hosted their Annual Meeting in San Francisco. During the event, the Standards Initiative for Pluripotent Stem Cell Research was launched, a new project that aims to provide recommendations and standards for the characterization of human pluripotent stem cell (hPSC) lines. Co-chaired by Dr. Tenneille Ludwig (WiCell Research Institute) and Prof. Peter Andrews (the University of Sheffield), four working groups were formed to address:
The half-day session consisted of presentations from the Steering Committee and the four Working Groups, as well as a lively and informative Q&A with the ~100 ISSCR delegates that were in attendance. The recommended guidelines for hPSC research standards are expected to be published by ISSCR later in 2022.
STEMCELL Technologies is an advocate for standardizing hPSC data reporting and quality control measures to limit experimental variability and ensure that relevant, reproducible findings are shared. We proudly sponsor the ISSCR Standards Initiative for Pluripotent Stem Cell Research as well as other societies and consortia that aim to set standards in the hPSC research field including the International Stem Cell Banking Initiative (ISCBI) and the Global Alliance for iPSC Therapy (GAiT). As such, it should come as no surprise that when developing our first induced pluripotent stem cell (iPSC) line for commercial use, SCTi003-A , we cut no corners to ensure that we not only meet, but exceed all of the recommended guidelines for hPSC characterization.
Continue reading this article to learn more about how SCTi003-A was sourced, developed, QC'd, and the extensive testing that has been performed to ensure you will be receiving one of the most highly characterized iPSC lines in the stem cell research field today.
To learn more about the ISSCR Standards Initiative, click here .
Cell Line Information
The Healthy Control Human iPSC Line, Female, SCTi003-A (Catalog no. 200-0511) was derived from peripheral blood mononuclear cells from a 48-year-old donor. Extensive quality control procedures were undertaken in the iPSC manufacturing process to ensure optimal product performance and reproducibility. SCTi003-A is karyotypically stable, demonstrates trilineage differentiation potential, expresses markers of the undifferentiated state, and was reprogrammed using a non-integrating reprogramming technology. The cell line may be used as a healthy control for a multitude of pluripotent stem cell research applications including downstream differentiation to lineage-specific cell types and organoids.
SCTi003-A is manufactured with mTeSR? Plus (Catalog no. 100-0276) and is fully compatible with STEMdiff? cell culture media products, allowing for standardized high-quality maintenance and differentiation to various cell types such as cardiomyocytes, neurons, astrocytes, and microglia.
SCTi003-A was derived from an αβ T cell and has undergone VDJ rearrangement.
Advantages:
Donor Information
STEMCELL collects donor demographic information ethically, using consent forms and protocols approved by either an Institutional Review Board (IRB), the Food and Drug Administration (FDA), the U.S. Department of Health and Human Services, and/or an equivalent regulatory authority. Donations are performed in the United States in compliance with applicable federal, state, and local laws, regulations, and guidance. Healthy donors must be over the age of 18, weigh at least 120 lb, have a body mass index (BMI) between 18.5 - 24.9, demonstrate no use of tobacco products, and be in good general health. Additionally, donors in our healthy pool are pre-screened using a health questionnaire aimed at excluding any donors with diseases, blood disorders, or other health concerns.?
Table 1 details attributes that were determined for the SCTi003-A donor. Age, diagnosis, ethnicity and/or race, and tobacco use were self-declared by the SCTi003-A donor. Sex, ancestry, height, weight, BMI, blood type, HLA haplotype, and pathogenic genetic variants were calculated using various methods detailed in the table legend.
Table 1. iPSC Line SCTi003-A Is Derived from a Healthy Female Donor. Demographic, health, and genetic characteristics of the SCTi003-A donor were compiled based on self-reported information and whole-exome sequencing. Sex was determined by karyotype. Ancestry was calculated by EthSEQ analysis from whole-exome sequencing data. HLA haplotype was determined by next-generation sequencing, sequence-base typing, and sequence-specific oligonucleotide probes as needed to obtain the required resolution. Other genetic variants were determined from whole-exome sequencing using ClinVar analysis. Blood type (ABO/Rh blood group) was determined by next-generation sequencing. Height, weight, and BMI were calculated at the donation facility.
Naming, Registration, and Certification of SCTi003-A by hPSCreg?
STEMCELL recognizes that a lack of well-defined guidelines for naming hPSC lines has led to confusion and duplication of cell line names and identities in the research field. As a result, we use the standardized nomenclature established by hPSCreg? that (1) unambiguously identifies a registered cell line, (2) allows tracing of subclones, and (3) enables the assignment of different cell lines to a particular donor. Table 2 demonstrates the hPSC line naming system that has been developed by hPSCreg? (Kurtz et al., 2018 ).
Table 2. Structure and Example of a Cell Line Name. hPSC line name components include the institution, cell line type, unique donor ID, and a letter that is incremented depending on how many cell lines are generated from the same donor. If a subclone has been generated, the name is extended using a hyphen and the subclone number starting with "1".
hPSCreg? is a global registry for PSC lines that aims to provide the community with a central and searchable hub and acts as a trustworthy data source by verifying ethical and biological conformity based on community standards. hPSCreg? has issued a certificate for SCTi003-A, indicating that evidence has been provided demonstrating that a minimal set of ethical and scientific standards have been met by the line. Ethical standards include documentation of informed consent procedures for the donation of the source material used for derivation of the iPSC line. Scientific standards include the documented testing of pluripotency using established and accepted assays. The hPSCreg? cell line certificate is essential for some funding agencies (e.g. the use of a hPSC line in research funded by the European Union).
To learn more about SCTi003-A on the hPSCreg? website, click here .
Quality Control
Extensive quality control procedures are implemented at every stage of STEMCELL’s iPSC manufacturing process (Table 3). Our commercial iPSC quality assessments and release criteria have been developed based on recommendations and guidance from the International Stem Cell Banking Initiative (ISCBI; 2009 ), the Global Alliance for iPSC Therapy (GAiT; 2018 ), and the consensus workshop hosted in June 2022 by the ISSCR Standards Initiative for Pluripotent Stem Cell Research . Continue reading to see the characterization data for SCTi003-A including morphology, chromosome analysis, copy number variants, pathogenic genetic variants, TP53 and BCOR status, donor ancestry, undifferentiated status, and pluripotency.
Table 3. Characterization Assessments Are Performed at Various Stages throughout the iPSC Manufacturing Process. Master cell banks are tested for identity, adventitious agents, genomic integrity and stability, survival, undifferentiated state, and pluripotency. Working cell banks and commercial vials are tested for a subset of these characterization criteria.
Morphology
Figure 1 demonstrates examples of the high quality undifferentiated cell morphology presented by the SCTi003-A cell line. Microscopy images were captured after thawing and expanding the cell line in mTeSR? Plus (Catalog no. 100-0276) on Matrigel?. The large, round iPSC colonies presented with defined borders, contained densely packed cells, and developed multi-layering in the center when the culture was ready to be passaged. The iPSCs retained prominent nucleoli and presented with a high nuclear-to-cytoplasmic ratio, as is expected for high quality undifferentiated hPSCs. We recommend that a culture is passaged once it has reached an optimal density consisting of large, multilayered colonies that appear as phase bright under a phase contrast microscope, and have just begun to merge.
Figure 1. SCTi003-A Human Pluripotent Stem Cells Demonstrate High-Quality Morphology in Routine Culture. Cryopreserved cells from line SCTi003-A were thawed and maintained in mTeSR? Plus on Corning? Matrigel? Matrix. (A) The resulting iPSC colonies have densely packed cells and show multi-layering when ready to be passaged. (B,C) Cells retain prominent nucleoli and high nuclear-to-cytoplasmic ratios.
Chromosome Analysis
Somatic mutations that generate a selective advantage, such as a greater propensity for self-renewal, can become fixed in hPSC cultures over time. This selection leads to the development of nonrandom genetic changes that are found in PSCs maintained for long periods in culture. These changes commonly involve non-random gains of chromosomes 12, 17, 20, and X, and are abnormalities that are also present in a number of human cancers. As a result, it’s important to karyotype PSC cultures regularly. The karyotype in Figure 2A was obtained from the SCTi003-A master cell bank at passage 26 and was characterized as “normal” with no clonal abnormalities detected. We perform a G-banded karyotype for every iPSC bank that we produce and include a lot-specific karyotype with the certificate of analysis that corresponds to the batch of cells that a commercial vial has been distributed from.
Human PSCs also frequently acquire a copy number variant (CNV), located at chromosome 20q11.21 upon prolonged culture. Once it has been gained, this CNV provides a growth advantage to PSCs as a result of resistance to apoptosis. Since this particular CNV is a genomic hallmark of some cancers, it represents a potential impediment to the use of PSCs in regenerative medicine. The shared region that’s common to all of these reported variants contains a dosage-sensitive antiapoptotic gene called BCL2L1, which has been identified as the driver gene in this mutation. The length of this duplicated region is small, ranging in size from between 0.6 to 4 Mb, meaning that this amplification is often missed by karyotyping. As a result, we use fluorescence in-situ hybridization (FISH), to look for any evidence of this mutation in our lines. We performed 20q FISH on the SCTi003-A master cell bank at passage 26. In this experiment, two probe signals were identified in 94% of 200 interphase cells examined for the 20p11.21 and 20q11.21 regions, indicating that there is no evidence for aneusomy of chromosome 20 in this iPSC line.?
Figure 2. SCTi003-A Human Pluripotent Stem Cells Maintain a Normal Karyotype. (A) G-T-L banding for thawed cells at p26 (n = 20) shows a normal karyotype with no evidence of clonal abnormalities at a band resolution of 450 - 550 G-bands per haploid genome. (B) Fluorescent in situ hybridization in a representative p26 iPSC using probes for 20p11 (green) and 20q11.21 (red). 94% of cells examined displayed two sets of two probe signals, indicating no aneusomy of chromosome 20 (n = 200).
Copy Number Variants
Copy number variants (CNVs) have the ability to affect cellular function or the interpretation of inherited variants. G-banding is the most widely used technique for monitoring the genomic stability of PSCs, however, this method only allows for the detection of large chromosomal rearrangements >5 Mb. As a result, SNP microarray technology can be used to increase the sensitivity of karyotyping by up to 50-fold. This assay detects aneuploidy, deletions, and duplications of represented loci, and regions of loss or absence of heterozygosity (LOH). The assay does not detect point mutations or balanced alterations, and that includes reciprocal translocations, Robertsonian translocations, inversions, and insertions.
Reportable copy number changes are gains or losses that are greater than 400 kb in size (rows highlighted in bold in Table 4). We identified two reportable copy number changes in the SCTi003-A master cell bank – a 0.476 Mb loss on chromosome 7 and a 0.634 Mb loss on chromosome 14. These changes at 7q34 and 14q11.2 are in T cell receptor regions, indicating that this is the result of VDJ recombination from T cell reprogramming. We performed the T cell clonality assay which identified that the parent cell lineage for the SCTi003-A cell line was an αβ T cell. As such, this SNP microarray result is in line with this finding.
Table 4. Single Nucleotide Polymorphism Microarray Analysis Characterizes SCTi003-A Copy Number Variants. DNA was extracted from a vial of SCTi003-A from the Master Cell Bank and subjected to SNP microarray analysis to identify large-scale CNVs. The cells display two reportable CNVs on chromosome 7 and 14. These losses are located in the TCR regions of the genome and are indicative of VDJ recombination process during T Cell development.
Pathogenic Genetic Variants
Whole exome sequencing of the SCTi003-A cell line was performed at a depth of 50x, indicating that each nucleotide of the exome (the collection of all coding sequences) is sequenced 50 times. From this redundant coverage, a highly accurate consensus sequence was derived, allowing for the identification of genomic SNPs. The resulting profile of genetic variants is compared against ClinVar , a public archive of reports that detail relationships between human genetic variants and phenotypes, and any resulting pathogenic or likely pathogenic variants were determined. ClinVar analysis includes germline and somatic variants of any size, type, or genomic location.
Five pathogenic or likely pathogenic variants were identified in the SCTi003-A cell line including mutations in AGXT, KLKB1, SLC12A3, NQO1, and RUNX1 (Table 5). Based on data from the 1000 Genomes Project , the average person has 18 pathogenic or likely pathogenic variants. All of the pathogenic or likely pathogenic variants identified in the SCTi003-A cell line can be cross-referenced to ClinVar using the ClinVar IDs found in the call table. As well as these variants, there were an additional 17 risk factors, 78 conflicting interpretations of pathogenicity, and 61 variants classified as uncertain significance. The whole exome sequencing data file for SCTi003-A is available separately for a small charge (Catalog no. 500-0261).
Table 5. Five Pathogenic or Likely Pathogenic Genetic Variants Were Identified in the SCTi003-A iPSC Line by Whole Exome Sequencing. DNA was purified from the SCTi003-A master cell bank and exon capture performed for coding regions and splice junction sites of 20,000 human genes, covering 60 Mb of DNA. Post-capture libraries were sequenced to a coverage of 50x using paired-end 150 nucleotide reads. Single Nucleotide Variants (SNVs) and insertions/deletions (indels) are detected across the entire exome after alignment to the GRCh38 human reference genome. Thresholding was performed to eliminate sequencing errors by only including regions covered by at least eight reads. In order to identify germline variants, heterozygous SNVs/indels were classified as regions in which minor alleles are represented by at least two unique reads and are present in at least 10% of all reads covering the locus. Resulting SNVs/indels were cross-referenced to ClinVar.
TP53 and BCOR Status
In 2017, Florian Merkle and colleagues reported that their whole-exome sequencing of 140 lines of human embryonic stem cells (26 of which were produced for clinical use), and 117 lines of iPSCs contained numerous mutations in TP53. Five unrelated lines of embryonic stem cells had six inactivating mutations in TP53 of the type commonly seen in cancers. Another nine mutations that are predicted to create errors in the DNA-binding domain of TP53 were found in iPSCs. Interestingly, the proportions of TP53-mutated cells increased with the number of passages in culture and rose particularly rapidly during some steps of differentiation. As such, it would seem that there is a strong selective advantage for TP53 mutations that reduce its protein function during reprogramming, during pluripotent-cell expansion, and during the differentiation of pluripotent cells and their derivatives in vitro. Separately to this, at the ISSCR Annual Meeting in 2017, Prof. Shinya Yamanaka presented findings of a point mutation in the tumor-suppressor gene BCOR in one iPSC line prepared for clinical use at CiRA in Japan, but the consequence of this mutation with respect to cell proliferation was not clear. Similar to TP53, the prevalence of BCOR variants was noted to increase markedly with the in vitro expansion of these iPSC lines. These two key findings have drawn attention to the acquisition of mutations in tumor suppressor genes in PSCs.
The status of TP53 and BCOR was analyzed for SCTi003-A by whole exome sequencing (81.08% and 73.89% of exons covered by 8+ reads respectively) and the resulting variants cross-checked using ClinVar (Table 6). No pathogenic or likely pathogenic variants were identified in TP53 and BCOR. Furthermore, no variants were identified in TP53 that were previously reported as common recurring mutations in human pluripotent stem cell cultures by Merkle et al. (2017) .
Table 6. No Pathogenic or Likely Pathogenic Genetic Variants Were Identified in TP53 or BCOR in the SCTi003-A iPSC Line by Whole Exome Sequencing. DNA was purified from the SCTi003-A master cell bank and exon capture performed for coding regions and splice junction sites of 20,000 human genes, covering 60 Mb of DNA. Post-capture libraries were sequenced to a coverage of 50x using paired-end 150 nucleotide reads. Single Nucleotide Variants (SNVs) and insertions/deletions (indels) are detected across the entire exome after alignment to the GRCh38 human reference genome. Thresholding was performed to eliminate sequencing errors by only including regions covered by at least eight reads. In order to identify germline variants, heterozygous SNVs/indels were classified as regions in which minor alleles are represented by at least two unique reads and are present in at least 10% of all reads covering the locus. Resulting SNVs/indels were cross-referenced to ClinVar. All TP53 and BCOR variants meeting these specifications were reported. For TP53, variants were cross-referenced to those reported by Merkle et al. (2017) .
Donor Ancestry
Analysis of the coding regions of the human genome for germline and somatic mutations by whole-exome sequencing is becoming commonplace in translational cancer genomics studies and in the field of precision medicine. Identifying a donor’s ancestry is essential for interpreting the impact of personal genetic variation on experimental results.
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We processed SCTi003-A whole exome sequencing data using EthSEQ , an analytical pipeline for identifying conserved ancestral groups. EthSEQ inspects sequencing data focusing on the differential SNP genotype profile containing variants known to be particularly informative of ancestral heritage. The donor profile was compared to a reference database containing thousands of genotypes of known ancestries collated from the 1000 Genomes Project . Subsequent principal component analysis (PCA) identified SCTi003-A ancestry by comparison to the reference database and calculated a percentage for each ancestry. STEMCELL’s internal ancestry determination workflow has been validated using whole exome sequencing data from the Simons Genome Diversity Project , a database that contains genotype data from hundreds of individuals with known ancestries, thus generating more comprehensive ancestral predictions.
The ancestry of SCTi003-A was determined to be closest to European with a contribution of 78.24% European and 21.76% South Asian (Figure 3).
Figure 3. SCTi003-A Underwent Ancestry Analysis Using EthSEQ. DNA was purified from the SCTi003-A master cell bank and exon capture performed for coding regions and splice junction sites of 20,000 human genes, covering 60 Mb of DNA. Post-capture libraries were sequenced to a coverage of 50x using paired-end 150 nucleotide reads. Single Nucleotide Variants (SNVs) and insertions/deletions (indels) are detected across the entire exome after alignment to the GRCh38 human reference genome. Ancestry was calculated using the EthSEQ R Package and the reference model, described by Romanel et al. (2017) . The reference model is generated based on genotype data that encompasses 123,024 loci from individuals with known ancestries, grouped into four major populations: African, European, South Asian, and East Asian. A corresponding ancestry is reported if it falls inside the ancestral group set (“Inside”). The nearest ancestry is reported if it falls outside the ancestral group set (“Closest”).
Undifferentiated Status
The undifferentiated status of the SCTi003-A cell line was analyzed using two separate methods. Firstly, expression of markers of the undifferentiated state, OCT4 and TRA-1-60, were analyzed by flow cytometry. Expression levels of 93.3% and 97.1% were achieved respectively for cells thawed from the master cell bank and passaged five times, exceeding our release criteria of >80% (Figure 4). Epi-Pluri-Score was also performed, an assay based on DNA methylation levels at three specific CpG sites. Two CpGs are located within the genes ANKRD46 (methylated in undifferentiated PSCs) and C14orf115 (non-methylated in undifferentiated PSCs), and a third is located within the pluripotency-associated gene OCT4. A positive Epi-Pluri-Score indicates undifferentiated status. SCTi003-A achieved an Epi-Pluri-Score of +21 which clusters the cell line with other undifferentiated pluripotent stem cells (Figure 5). This data consists of 2,215 independent DNA methylation profiles, demonstrating 99.9% specificity and 98.9% sensitivity.
Figure 4. SCTi003-A iPSCs Express Undifferentiated Cell Markers. Cell line SCTi003-A was characterized using flow cytometry for undifferentiated cell markers OCT3/4 and TRA-1-60. (A) Percentage marker expression was quantified five passages after thawing from the master cell bank from analyses of three technical replicates. Representative flow cytometry plots are displayed for (B) TRA-1-60 and (C) OCT3/4.
Figure 5. SCTi003-A Undifferentiated Status was Assessed by Epi-Pluri-Score. DNA-methylation was analyzed at three specific CpG sites. One of these CpGs was localized within the pluripotency-associated gene POU5F1 (also known as OCT4). The difference in DNAm levels (β-values) of CpGs in ANKRD46 and C14orf115 was determined and combined as Epi-Pluri-Score. The red and blue clouds refer to DNAm levels measured by pyrosequencing of 163 undifferentiated and 46 differentiated cell preparations, respectively. Epi-Pluri-Score classified the SCTi003 A cell line as undifferentiated (positive Epi-Pluri-Score).
Pluripotency
Pluripotency of the SCTi003-A cell line was assessed by in vitro directed trilineage differentiation using the STEMdiff? Trilineage Differentiation Kit (Catalog no. 05230). Expression levels of lineage-specific markers were assessed by flow cytometry following five days of culture for endoderm and mesoderm lineages, and following seven days of culture for the ectoderm lineage. Two markers from each embryonic germ layer were assessed including PAX6 and NESTIN for ectoderm, NCAM and BRACHYURY for mesoderm, and CXCR4 and SOX17 for endoderm. For the SCTi003-A master cell bank, all lineage-specific markers were expressed by >80% of differentiated cells, exceeding our release criteria of >70%. This result is consistent with the pluripotent state.
Figure 6. SCTi003-A Human Pluripotent Stem Cells Demonstrate a High Trilineage Differentiation Capacity. Cells from SCTi003-A were split into three groups, differentiated using the STEMdiff? Trilineage Differentiation Kit, and then subjected to flow cytometry analysis. Two markers for each embryonic germ layer were assessed, and bars present mean marker expression for each group of cells (n = 2 biological replicates).
Certificate of Analysis
The SCTi003-A Certificate of Analysis includes information relating to the product, cell line, recommended culture conditions, donor information, and detailed results for morphology, viability and recovery, cell line identity, sterility testing, mycoplasma testing, viral screening, parent cell lineage determination, chromosome analysis, 20q status, copy number variants, donor ancestry, genetic variants, TP53 and BCOR status, undifferentiated status, and pluripotency.
The certificate of analysis for SCTi003-A Lot # 2205404000 can be found here .
Differentiation
SCTi003-A has been optimized for compatibility with TeSR? media for maintenance and STEMdiff? for differentiation. To date, we have tested the compatibility of SCTi003-A with over 15 differentiation protocols including differentiation to neural progenitor cells, microglia, forebrain neurons, midbrain neurons, astrocytes, neural crest cells, sensory neurons, motor neurons, cerebral organoids, choroid plexus organoids, dorsal forebrain organoids, atrial and ventricular cardiomyocytes, hematopoietic progenitor cells, endothelial cells, and intestinal organoids. Continue reading to see the differentiation data for a subset of these cell types.
Neural Progenitor Cells
Neural progenitor cells (NPCs) were generated from SCTi003-A using the STEMdiff? SMADi Neural Induction Kit (Catalog no. 08581) monolayer protocol. The resulting cells were cryopreserved and subsequently thawed, re-established in culture, and fixed for immunocytochemistry analysis. NPC markers PAX6 and SOX1 were expressed by over 90% of cells in the culture and class III β-tubulin (TUJ1; a marker for mature neurons) demonstrated low expression, indicative of a neural progenitor-like population (Figure 7 A, B, C, E). The resulting NPCs presented with a small, teardrop-shaped morphology that is expected for this cell type (Figure 7 D).
Figure 7. SCTi003-A Human Pluripotent Stem Cells Can Efficiently Differentiate into Neural Progenitor Cells. Expression was quantified for (A) PAX6, (B) SOX1, and (C) class III β-tubulin (TUJ1). (D) NPCs displayed the expected small, teardrop-shaped morphology. (E) Marker expression was quantified for NPC markers and mature neuronal markers. Error bars represent standard deviation (n = 2 biological replicates).
Cardiomyocytes
Cardiomyocytes were generated using the STEMdiff? Ventricular Cardiomyocyte Kit (Catalog no. 05010), and the monolayers at Day 15 exhibited a beating behavior (Figure 8 A-B). cTNT, a marker specific for cardiomyocytes, was expressed by 89.5% of the cell population (Figure 8 C), and when the cells were re-plated on a microelectrode array (MEA) for functional analysis, a beat rate of 25 bpm was recorded with a field potential of approximately 500 ms (Figure 8 D-E). These numbers are expected for ventricular cardiomyocytes, usually ranging from between 25 to 50 bpm. This range is lower than atrial cells as the inward calcium transient is more pronounced, prolonging the action potential, meaning that the next beat can’t happen until that process is complete.
Figure 8. SCTi003-A Human Pluripotent Stem Cells Can Successfully Differentiate into Ventricular Cardiomyocytes. (A) Monolayer cultures at Day 15. (B) Beating ventricular cardiomyocytes can be replated and maintained in a well of an MEA plate for functional analysis. (C) cTnT expression was detected by flow cytometry. (D) Beat rate (n = 3 replicates, mean +/-SD plotted). (E) Field potential duration (n = 3 replicates, mean +/-SD plotted), as assessed by MEA.
Hematopoietic Progenitor Cells
The STEMdiff? Hematopoietic Kit (Catalog no. 05310) was used to generate hematopoietic progenitor cells (HPCs) from the SCTi003-A cell line, producing dual-positive CD34+/CD45+ cells with an efficiency of >40% and a yield of over 80,000 cells per cm2 (Figure 9 A). Bright field images of SCTi003-A-derived HPCs (Figure 9 B) indicate that the cells transitioned through a typical endothelial-to-hematopoietic transition (EHT). Resulting HPCs were subjected to the colony colony-forming unit (CFU) assay with MethoCult? SF H4636 (Catalog no. 04636) and incubated for 14 days (Figure 9 C). Myeloid and erythroid colonies were identified with a skew towards the myeloid lineage (Figure 9 D). This is typical of a PSC line that produces a high yield of HPCs, whereas low proliferative lines tend to have higher erythroid, or even lymphoid potential.
Figure 9. SCTi003-A iPSCs Can Form Hematopoietic Progenitor Cells and Have Colony-Forming Potential. (A) Percentages and yields of CD34+CD45+ HPCs per cm2 after differentiation (n = 2 biological replicates). (B) Bright field image of SCTi003-A-derived HPCs. Resulting HPCs were subjected to the CFU assay (n = 2). (C) After 14 days of incubation, colonies were imaged with STEMvision? and (D) enumerated from digital images.
Microglia
Once SCTi003-A-derived HPCs were generated, the cells were cryopreserved, thawed, and subsequently differentiated to microglia using the STEMdiff? Microglia Differentiation and Maturation Kits (Catalog no. 100-0019 and 100-0020). The resulting cells were small with visible processes, non-adherent on Matrigel?, and exhibited a small nuclear-to-cytoplasm ratio (Figure 10 A). Dual expression of CD45+ and CD11b+ was analyzed on Day 27 of differentiation and 98.7% of cells were positive for these markers (Figure 10 B). The resulting cells were also adherent on poly-D-lysine and contained less than 20% monocyte-like cells as assessed by May-Grunwald stain. These are cells that are large with a slightly stained cytoplasm, as pointed out by the white arrow (Figure 10 C).
Figure 10. SCTi003-A Human Pluripotent Stem Cells Can Effectively Differentiate into Microglia. (A) Bright field image of SCTi003-A-derived microglia. (B) Co-expression of CD45 and CD11b observed by flow cytometry. (C) The resulting cells are also adherent on poly-D-lysine, and contain < 20% monocyte-like cells (large, with lightly stained cytoplasm; arrow) as assessed by May-Grunwald Giemsa stain at Day 27.
Dorsal Forebrain Organoids
As well as 2-dimensional differentiation, the SCTi003-A cell line was also differentiated in 3D to organoids. Neural organoids were generated using the STEMdiff? Dorsal Forebrain Organoid Differentiation Kit (Catalog no. 08620). These structures were maintained until Day 105 with the STEMdiff? Neural Organoid Maintenance Kit (Catalog no. 100-0120) before fixing, cryosectioning, and immunofluorescent staining. The resulting dorsal forebrain organoids were stained for DAPI, MAP2, NEUN, and GFAP (Figure 11).
Figure 11. SCTi003-A Human Pluripotent Stem Cells Can Successfully Differentiate into Neural Organoids. Neural organoids were stained for (A) DAPI (blue), (B) MAP2 (magenta), (C) NEUN (yellow), and (D) GFAP (cyan). (E) 4-channel merged image. Panels show cellular-level detail at 63x magnification. Insets show the full cryosection at 10x magnification.
Intestinal Organoids
SCTi003-A successfully differentiated into intestinal organoids using the STEMdiff? Intestinal Organoid Kit (Catalog no. 05140). Figure 12 A-F demonstrates the cells transitioning between varying developmental stages starting from plated iPSCs at Day 0, uniform monolayers displaying characteristics of patterning to definitive endoderm at Day 3, visible mid-/hindgut 3D structures atop the monolayer culture at Day 4, detachment of these spheroids from the mid-/hindgut culture at Day 7, collections of spheroids embedded into Matrigel? domes for subsequent differentiation into intestinal organoids at Day 8, and significant expansion of these organoids using STEMdiff? Intestinal Organoid Growth Medium (Catalog no. 05145) after just six days in matrix at Day 13.
Figure 12. Human Pluripotent Stem Cells from Line SCTi003-A Can Successfully Differentiate into Intestinal Organoids. (A) Plated iPSCs, (B) definitive endoderm, (C) mid-/hindgut 3D structures, (D) detachment of spheroids, (E) embedded spheroids, and (F) expanded intestinal organoids.
Scale-Up In Bioreactors
We tested the ability of SCTi003-A to scale-up in bioreactors using TeSR? 3D. In order to initiate this experiment, SCTi003-A cells were transitioned from 2D culture in mTeSR? Plus (Catalog no. 100-0276) to suspension culture for eight serial passages in TeSR? 3D with consistent expansion. Initially, cells were expanded using a 250 mL Nalgene Storage Bottle with media volumes ranging between 15 - 60 mL. The cells were further expanded for an additional three passages using the PBS-MINI MagDrive Bioreactor (Catalog no. 100-1005 and 100-1006) with a suspension culture volume of 100 mL. The gentle yet efficient mixing provided by the Vertical-Wheel? impeller enabled expansion of SCTi003-A (shear-sensitive cells) without anti-foaming agents or shear protectants. The linear LOG cumulative fold expansion plot indicates robust and consistent expansion with no lag or adaptation phase at the beginning of culture when the cells transitioned from a 2D to a 3D environment (Figure 13 A). SCTi003-A obtained an average daily fold expansion of 1.5x with each passage being 3 - 4 days long. A total of 1 billion viable cells was obtained after 5 passages. Cells demonstrated a morphology characteristic of hPSC aggregates in suspension culture including spherical shape, clear edges (not smooth or shiny), even color, and a dimpled or pock-marked appearance.
Figure 13. SCTi003-A Human Pluripotent Stem Cells Demonstrate Efficient Scale-Up in Bioreactors. (A) LOG cumulative fold expansion plot over eight passages following transition from 2D maintenance to 3D expansion. (B) Bright field microscopy image of 3D aggregates.
Summary
STEMCELL Technologies has developed the Healthy Control Human iPSC Line, Female, SCTi003-A (Catalog no. 200-0511) for academic and/or commercial purposes, with the most stringent ethical, characterization, and quality standards in mind.
SCTi003-A is available now at a price of $1500 USD per vial. Annual license fees may apply. To request a quote, click here .
For more information about STEMCELL's iPSC lines, refer to our Frequently Asked Questions on iPSCs .
For any other queries, click here to contact STEMCELL's iPSC Team or email us at [email protected]