Characterization of induced pluripotent stem cell lines from patients of African American ancestry

Abstract

Induced pluripotent stem cells (iPSCs) are a valuable platform for studying human biology and developing patient-specific cellular models. However, individuals of African American ancestry remain underrepresented in existing iPSC repositories, limiting the diversity of available research resources. To address this gap, we generated and characterized two iPSC lines derived from healthy donors of African American ancestry. Both lines exhibited normal morphology, expression of pluripotency markers, trilineage differentiation potential, stable karyotypes, and absence of mycoplasma contamination. Short tandem repeat analysis confirmed donor identity. These well-characterized iPSC lines provide a valuable resource for future studies investigating ancestry-specific genetic and cellular mechanisms relevant to human disease.

Resource table

Unique stem cell line identifier	SCVIi156-A SCVIi157-A
Alternative stem cell identifier	SCVIi156-A SCVIi157-A
Institution	Stanford University
Contact information of distributor	Joseph C. Wu, joewu@stanford.edu
Type of cell lines	iPSC
Origin	Human
Additional origin info required for human ESC or iPSC	SCVIi156-A; Age: 58; Sex: Male; Ethnicity: African American SCVi157-A; Age: 50; Sex: Female; Ethnicity: African American
Cell source	Peripheral blood mononuclear cells (PBMCs)
Clonality	Clonal
Method of reprogramming	Integration-free Sendai virus expressing human OCT4, SOX2, KLF4, and c-MYC
Associated disease	No associated disease. Healthy cell lines from donor patients.
Gene/locus	N/A N/A
Date archived/stock date	SCVIi156-A: 07/15/2024 SCVIi157-A: 08/08/2024
Cell line repository/bank	https://hpscreg.eu/cell-line/SCVIi156-A https://hpscreg.eu/cell-line/SCVIi157-A
Ethical approval	The generation of the lines was approved by the Administrative Panel on Human Subjects Research (IRB) under IRB #29904 “Derivation of Human Induced Pluripotent Stem Cells (Biorepository).”

2. Resource utility

Individuals of African American ancestry remain underrepresented in existing induced pluripotent stem cell (iPSC) repositories despite the importance of diverse donor representation for biomedical research. We report the generation and characterization of two iPSC lines derived from healthy donors of African American ancestry. These lines provide a well-characterized cellular resource that may facilitate future investigations into ancestry-specific genetic variation, cellular biology, and mechanisms relevant to human health and disease (Wu, 2026).

3. Resource detail

iPSCs have become an important platform for studying human biology and generating patient-derived cellular resources for disease modeling, drug discovery, and efficacy testing (Liu, 2026) (Yildirim, 2025). Because iPSCs retain the genetic background of the donor and possess the capacity for self-renewal and differentiation, they provide a renewable source of human cells for basic and translational research. In addition, iPSC technology overcomes several limitations associated with animal models and primary human tissues by providing an accessible and genetically defined in vitro system (Davidson, 2025). The establishment of well-characterized iPSC lines from diverse populations is therefore important for expanding the availability of representative cellular resources for biomedical research.

To contribute to this effort, we generated two iPSC lines from healthy donors of African American ancestry. Line SCVIi156-A was derived from a 58-year-old male donor, and line SCVIi157-A was derived from a 50-year-old female donor (Table 1). Peripheral blood mononuclear cells (PBMCs) were isolated from donor blood samples and reprogrammed using integration-free Sendai viral vectors expressing the four Yamanaka factors OCT4, SOX2, KLF4, and c-MYC. Both resulting iPSC lines displayed morphological characteristics consistent with pluripotent stem cells (Fig. 1A). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) demonstrated robust expression of the pluripotency-associated genes NANOG and SOX2 in both lines (Fig. 1B). Sendai viral sequences were detected at early passages but were absent by passage 30, confirming successful clearance of the reprogramming vectors and generation of transgene-free iPSC lines (Fig. 1C).

Table 1.

Characterization and validation.

Classification	Test	Result	Data
Morphology	Photography brightfield	Visual record of the line: normal	Fig. 1A
Phenotype	Quantitative analysis: RT-qPCR Qualitative analysis: immunofluorescent staining	NANOG and SOX2 are expressed in all iPSC lines and absent in differentiated iPSC-CMs Positive expression of pluripotency markers: NANOG, OCT3/4, SOX2	Fig. 1B Fig. 1D
Genotype	Karyotype (G-banding) and resolution	KaryoStat™ Assay, resolution 1–2 Mb: Normal karyotype: 46, XY for 156 and 46, XX for 157	Fig. 1F
Identity	Microsatellite PCR (mPCR) STR analysis	N/A 16 loci tested and matched well	N/A Available with authors
Microbiology and virology	Mycoplasma	Mycoplasma testing by luminescence: negative	Fig. 1G
Differentiation potential	Directed differentiation	Positive IF staining of three germ layer markers	Fig. 1E
List of recommended germ layer markers	Expression of these markers has to be demonstrated at mRNA or protein (IF) levels, at least 2 markers need to be shown per germ layer	Ectoderm: PAX6, OTX2 Endoderm: SOX17, FOXA2 Mesoderm: TBX6, BRACHYURY	Fig. 1E

Fig. 1.

Generation of iPSC lines from African American patients.

Immunofluorescence staining further confirmed expression of the pluripotency markers OCT3/4, NANOG, and SOX2 in both cell lines (Fig. 1D). To assess pluripotency, directed differentiation experiments were performed and demonstrated the ability of both lines to generate derivatives of all three germ layers, including ectoderm (PAX6, OTX2), endoderm (SOX17, FOXA2), and mesoderm (TBX6, BRACHYURY) (Fig. 1E). Genomic stability was evaluated using the KaryoStat^™ whole-genome assay. No pathogenic copy number variations, regions of loss of heterozygosity, or other detectable chromosomal abnormalities were identified in either line. Both lines demonstrated normal karyotypes, consistent with 46,XY for SCVIi156-A and 46,XX for SCVIi157-A (Fig. 1F). Mycoplasma testing using a bioluminescence-based detection assay confirmed that both iPSC lines were free of detectable mycoplasma contamination (Fig. 1G). Cell line identity was further verified by short tandem repeat (STR) analysis, which demonstrated concordance across 16 loci between each donor PBMC sample and its corresponding iPSC line (data available upon request from the authors).

4. Materials and methods

4.1. Reprogramming

PBMCs were isolated from donor blood using Percoll density gradient medium (GE Healthcare, Cat #17089109) and washed three times with phosphate-buffered saline (PBS; Thermo Fisher Scientific, Cat #141190144). Isolated PBMCs were cultured in StemPro-34 SFM medium (Thermo Fisher Scientific, Cat #10639011) supplemented with 100 ng/mL FLT3 (Thermo Fisher Scientific, Cat #PHC9414), 100 ng/mL stem cell factor (SCF; PeproTech, Cat #300-07), 20 ng/mL interleukin-6 (IL-6; Thermo Fisher Scientific, Cat #PHC0063), 20 ng/mL interleukin-3 (IL-3; PeproTech, Cat #200-3), and 20 ng/mL erythropoietin (EPO; Thermo Fisher Scientific, Cat #PHC9631).

Approximately 3 × 10^5 PBMCs were reprogrammed using the CytoTune^™-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, Cat #A16517), which delivers OCT4, SOX2, KLF4, and c-MYC. Reprogrammed cells were plated onto Matrigel-coated culture plates (Corning, Cat #356231; 1:500 dilution) and maintained in StemPro-34 medium. Seven days after transduction, cultures were transitioned to StemMACS^™ iPS-Brew XF medium (Miltenyi Biotec, Cat #130-104-368) supplemented according to the manufacturer’s instructions (Miltenyi Biotec, Cat #130-107-087). Emerging iPSC colonies were manually picked between days 10 and 15, expanded, and cryopreserved (Wu, 2026).

4.2. Cell culture

iPSCs were maintained in complete StemMACS^™ iPS-Brew XF medium. During passaging, 10 μM ROCK inhibitor Y-27632 (Selleck Chemicals, Cat #S1049) was added to enhance cell survival. Cells were passaged using 0.5 mM EDTA (Invitrogen, Cat #15575-038) upon reaching approximately 85% confluency and replated onto Matrigel-coated plates at split ratios ranging from 1:6 to 1:12. Cultures were maintained at 37°C in a humidified incubator containing 5% CO₂.

4.3. RNA extraction and RT-qPCR

Expression of pluripotency-associated genes (NANOG and SOX2) and Sendai viral sequences was assessed at passage 22. Total RNA was extracted using Trizol^™ Reagent (Thermo Fisher Scientific, Cat #15596026) and purified using the Direct-zol RNA Microprep Kit (Zymo Research, Cat #R2062). Complementary DNA (cDNA) was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad, Cat #1708891). Quantitative PCR was performed using TaqMan assays listed in Table 2 according to the manufacturer’s instructions.

Table 2.

Details of reagents.

Antibodies used for immunocytochemistry/flow-cytometry
	Antibody	Dilution	Company Cat #RRID	RRID
Pluripotency marker	Mouse IgG1κ anti-SOX 2	1:200	Santa Cruz Biotechnology Cat #sc-365823	RRID: AB_10842165
Pluripotency marker	Mouse IgG2bκ anti-Oct-3/4	1:200	Santa Cruz Biotechnology Cat #sc-5279	RRID: AB_628051
Pluripotency marker	Rabbit anti-NANOG	1:200	Proteintech Cat #142951–1-AP	RRID: AB_1607719
Ectoderm marker	Goat anti-OTX2	1:200	R&D Systems Cat #963273	RRID: AB_2157172
Ectoderm marker	Rabbit anti-PAX6	1:200	Thermo Fisher Scientific Cat #42–6600	RRID: AB_2533534
Endoderm marker	Goat anti-SOX17	1:200	R&D Systems Cat #AF1924	RRID: AB_355060
Endoderm marker	Rabbit anti-FOXA2	1:200	Thermo Fisher Scientific Cat #701698	RRID: AB_2576439
Mesoderm marker	Goat anti-BRACHYURY	1:200	R&D Systems Cat #963427	RRID: AB_2200235
Mesoderm marker	Rabbit anti-TBX6	1:200	Thermo Fisher Scientific Cat #PA5-35102	RRID: AB_2552412
Secondary Antibody	Alexa Fluor 488 Goat anti-Mouse IgG1	1:1000	Thermo Fisher Scientific Cat #A-21121	RRID: AB_2535764
Secondary Antibody	Alexa Fluor 647 Goat anti-Mouse IgG2b	1:500	Thermo Fisher Scientific Cat #A-21242	RRID: AB_2535811
Secondary Antibody	Alexa Fluor 555 Goat anti-Rabbit IgG (H + L)	1:500	Thermo Fisher Scientific Cat #A-21428	RRID: AB_141784
Secondary Antibody	Alexa Fluor 488 Donkey anti-Goat IgG (H + L)	1:1000	Thermo Fisher Scientific Cat #A-11055	RRID: AB_2134018
Secondary Antibody	Alexa Fluor 555 Donkey anti-Rabbit IgG (H + L)	1:500	Thermo Fisher Scientific Cat #A-31572	RRID: AB_2180682

Primers
	Target	Size of Band	Forward/Reverse primer (5′-3′)
Sendai virus plasmids (qPCR)	Sendai virus genome	181	Mr04269880_mr
Housekeeping gene (qPCR)	GAPDH	471	Hs02786624_g1
Pluripotency marker (qPCR)	SOX2	258	Hs04234836_s1
Pluripotency marker (qPCR)	NANOG	327	Hs02387400_g1

4.4. Immunofluorescence staining

Immunofluorescence staining was performed at passage 21 to assess pluripotency marker expression. Cells were fixed in 4% formalin (Sigma-Aldrich, Cat #HT501128) for 10 min at room temperature and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Cat #9036-19-5). Following PBS washes, cells were blocked in 5% goat serum prepared in PBST for 30 min at room temperature. Primary antibodies (Table 2) were applied overnight at 4°C. Following washing, appropriate secondary antibodies (Table 2) were applied for 30 min at room temperature. Nuclei were counterstained using NucBlue Fixed Cell ReadyProbes Reagent (Thermo Fisher Scientific, Cat #R37606). Images were acquired using a Keyence BZ-X800 microscope.

4.5. Trilineage differentiation

Pluripotency was evaluated by directed differentiation into ectoderm, mesoderm, and endoderm lineages. Ectoderm differentiation was performed using the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems, Cat #SC027B). Mesoderm differentiation was induced using RPMI medium supplemented with B27 minus insulin (Gibco, Cat #11875-085 and #A18956-01) and 6 μM CHIR99021 (Selleck Chemicals, Cat #S2924) for 48 h. Endoderm differentiation was performed using the StemDiff^™ Definitive Endoderm Differentiation Kit (STEMCELL Technologies, Cat #05110). Lineage specification was confirmed by immunostaining for established germ layer markers.

4.6. Sequencing

Genomic DNA was amplified using Quick-Load Taq 2X Master Mix (New England Biolabs, Cat #M0271S) and primers listed in Table 2. PCR products were generated using the following cycling conditions: 95°C for 3 min; 39 cycles of 95°C for 30 sec, 58°C for 15 sec, and 68°C for 30 sec; followed by a final extension at 68°C for 5 min. PCR products were analyzed by Sanger sequencing through Quintara Biosciences.

4.7. Karyotyping

Genomic integrity was assessed at passage 12 using the KaryoStat^™ Assay (Thermo Fisher Scientific), a genome-wide SNP array designed to detect copy number variations (CNVs) and regions of loss of heterozygosity (LOH). Data were analyzed using Chromosome Analysis Suite (ChAS) software. Karyotypes were considered normal in the absence of pathogenic CNVs, LOH events, or structural chromosomal abnormalities. Both lines demonstrated normal karyotypes consistent with 46,XY (SCVIi156-A) and 46,XX (SCVIi157-A).

4.8. Short tandem repeat analysis

Genomic DNA was isolated from donor PBMCs and corresponding iPSC lines at passage 22 using the DNeasy Blood & Tissue Kit (Qiagen, Cat #69504). STR profiling was performed using the CLA IdentiFiler^™ Direct PCR Amplification Kit (Thermo Fisher Scientific, Cat #A44661), which evaluates 15 STR loci and the amelogenin sex-determination marker. Fragment analysis was performed by capillary electrophoresis at the Stanford Protein Nucleic Acid Facility using an ABI3130xl Genetic Analyzer. Donor identity was confirmed by concordance across all analyzed loci between PBMC and iPSC samples (data available upon request from the authors).

4.9. Mycoplasma detection

Mycoplasma contamination was assessed using the MycoAlert^™ Mycoplasma Detection Kit (Lonza, Cat #LT07-318) according to the manufacturer’s instructions. This bioluminescence-based assay measures mycoplasma-associated enzymatic activity in culture supernatants. Results were expressed as the ratio of luminescence measurements obtained before and after substrate addition. Samples with ratios below the manufacturer-recommended threshold of 1.0 were considered negative for mycoplasma contamination (this threshold is indicated by the dashed line in Fig. 1G). As shown, the negative control (A/B = 0.75) and positive control (A/B = 1.33) confirm assay validity. Both iPSC lines returned ratios below the threshold of 1.0, with SCVIi156-A returning A/B = 0.83 and SCVIi157-A returning A/B = 0.69, confirming negative contamination.

Data availability

Data will be made available on request.