Generation and characterization of POMC-tdTomato reporter human pluripotent stem cell lines

Abstract

Proopiomelanocortin (POMC) is a precursor polypeptide that undergoes extensive, tissue-specific post-translational processing. It is expressed in several tissues, including pituitary gland, hypothalamus, brain stem, and skin. The hypothalamic POMC neurons in the arcuate nucleus are major neuronal populations involved in the regulation of body weight. In these neurons, POMC is processed into several peptides, among them the anorexigenic alpha-melanocyte stimulating hormone. Thus, the POMC neurons in the ARC have been named “satiety” neurons and are highly desirable drug targets. Here, we performed CRISPR/Cas9-mediated insertion of tdTomato reporter at the endogenous POMC locus, enabling direct visualization of POMC expression through tdTomato fluorescence in human pluripotent stem cell (hPSC)-derived hypothalamic neurons. This reporter line enables real-time visualization of POMC neuron differentiation, and selective enrichment of these populations for molecular, functional, and pharmacological studies. This line is readily available as new alternative method (NAM) platform, to support disease modeling and drug discovery in metabolic and neuroendocrine disorders within a human context.

1. Resource utility

These WA09 POMC-tdTomato human PSC reporter lines will serve as a resource for studies involving the differentiation of hPSCs into POMC expressing cell types. We demonstrate differentiation of these reporter cells into hypothalamic arcuate POMC neurons, validate reporter expression and their utility for functional characterization of these POMC neurons (see Table 1.).

Table 1.

Characterization and validation.

Classification	Test	Result	Data
Morphology	Microscopy Bright-field	Normal	Fig. 1 panel E Suppl. Fig. 1 panel A
Phenotype	Qualitative analysis (Immunocytochemistry)	OCT4, NANOG, SOX2	Fig. 1 panel E Suppl. Fig. 1 panel A
	Quantitative analysis (RT-qPCR)	Oct4, Nanog, Sox2 gene expression	Fig. 1 panel D
Genotype	Karyotype (CGH array) and resolution	46, XX Resolution: 50–100 kb	Supplemental
mtDNA analysis (IF APPLICABLE)	N/A	N/A
Identity		Performed
	STR analysis	24 sites tested; matched	Submitted in archive with journal.
Mutation analysis (IF APPLICABLE)	Sequencing (Sanger)	Homozygous and Heterozygous	Fig. 1 panel F Suppl. Fig. 1 panel B
	Southern Blot OR WGS	N/A
	Off-target nuclease analysis with mismatches	No Mutations found	Suppl. Fig. 1 panel C
Microbiology and virology	Mycoplasma	Mycoplasma testing by luminescence. Negative	Suppl. Fig. 1 panel D
Differentiation potential	Directed differentiation	Ectoderm (PAX6), mesoderm (TBXT), endoderm (SOX17), hypothalamic neurons (POMC)	Fig. 1 panel G Fig. 1 panel H
Donor screening (OPTIONAL)	HIV 1 + 2 Hepatitis B, Hepatitis C	N/A	N/A
Genotype additional info (OPTIONAL)	Blood group genotyping	N/A	N/A
Genotype additional info (OPTIONAL)	HLA tissue typing	N/A	N/A

2. Resource details

The POMC gene encodes the precursor protein proopiomelanocortin, which undergoes enzymatic cleavage to generate multiple biologically active peptides, most notably adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormones (MSHs), and β-endorphin. These peptides regulate diverse physiological processes, including energy homeostasis, stress and inflammatory responses, pigmentation, and mood modulation (Quarta et al., 2021). Within the hypothalamus, POMC is prominently expressed in the POMC neurons of the arcuate nucleus mediating satiety.

In this study, we generated a POMC knock-in reporter line by CRISPR/Cas9-mediated insertion of tdTomato into the endogenous POMC locus (Fig. 1A). Following nucleofection using a pCas9_CD4 (Stratigopoulos et al., 2018) and POMC-P2A-tdTomato targeting vector, cells were plated for recovery. Subsequently, cells were enriched using CD4 beads magnetic purification and plated at clonal density. Single colony picking yielded five independent clonal lines three carrying tdTomato (1G, 1B2, and 10E; Fig. 1B) and two non-edited clones (2A, and 8A; Fig. 1B). PCR using three primer sets (Table 2) was confirmed locus-specific insertion in clones 1G, 1B2 and 10E (Fig. 1C; primer set 1 and 2) and identify homozygous and heterozygous clones 1G, 1B2, and 10E, respectively (Fig. 1C; primer set 3). Clones with validated insertion were further analyzed by RT-qPCR to assess expression of pluripotency markers OCT4, SOX2, and NANOG, normalized to the parental WA09 line and compared against ectoderm-differentiated WA09 as a negative control (Fig. 1D).

Fig. 1. Generation and validation of a POMC-tdTomato knock-in reporter line.

(A) Schematic of CRISPR/Cas9-mediated knock-in strategy for insertion of a tdTomato reporter into the endogenous POMC locus. Primer sets 1–3 are positioned outside the RHA or LHA and within tdTomato where appropriate, as indicated. (B) Transfection of WA09 parental line followed by expansion, single-cell sorting and clonal expansion yielded five independent clonal lines (1G, 1B2, 2A, 8A, and 10E). (C) PCR genotyping using three primer sets confirmed locus-specific insertion and distinguished homozygous (1G, 1B2) from heterozygous (10E) knock-in clones. (D) RT-qPCR analysis of pluripotency markers OCT4, SOX2, and NANOG in validated clones, normalized to the parental WA09 line and compared against ectoderm-differentiated WA09 as a negative control. (E) Immunocytochemistry confirming OCT4, SOX2, and NANOG protein expression in homozygous (1G) clone. (F) Sanger sequencing verifying precise tdTomato reporter integration at the POMC locus. (G) Immunostaining of directly differentiated cultures of clone 1G into three germ layers showing expression of lineage-specific markers: PAX6 (ectoderm), TBXT (mesoderm), and SOX17 (endoderm). (H) Live-cell imaging (Incucyte) of 1G POMC-tdTomato clone at day 21 of hypothalamic neuronal differentiation showing robust tdTomato fluorescence. (I) Representative spontaneous action potential firing of POMC-tdTommato (W09 1G clone)-expressing hypothalamic neuron at −50 mV.

Table 2.

Reagents details.

	Antibodies used for immunocytochemistry/flow-cytometry
	Antibody	Dilution	Company Cat #	RRID
Pluripotency Undifferentiation Markers	Mouse anti-OCT4	1:100	Santa Cruz Cat# SC5279	AB_628051
	Goat anti-SOX2	1:100	R&D Cat# AF2018	AB_355110
	Rabbit anti-NANOG	1:200	Cell Signaling Technology Cat# 4903	AB_10559205
Differentiation Markers	Rabbit anti-PAX6	1:100 1:100	Biolegend Cat# 901,302	AB_2749901
	Rabbit anti-TBXT	1:1000	Cell Signaling Technology Cat# 81,694	AB_2799983
	Rabbit anti-SOX17		Cell Signaling Technology Cat# 81,778	AB_2650582
Secondary antibodies	Donkey anti-mouse Alexa Fluor 568	1:1000	Thermo Fisher Scientific Cat# A10037, A32849, A21206	AB_11180865
	Donkey anti-goat Alexa Fluor 647			AB_2762840
	Donkey anti-rabbit Alexa Fluor 488			AB_2535792
	Primers
	Target	Size of band	Forward/Reverse primer (5′–3′)
Sequencing Primer set 1	tdTomato insertion	200 bp	GTAAAACGACGGCC
			AGTCGCTACGGCGGT
			TTCAT/GGAAACAGC
			TATGACCATGATGA
			ACTCTTTGATGACC
			TCCT
Sequencing Primer set 2	tdTomato insertion	314 bp	GTAAAACGACGGC
			CAGTCACCACCTGT
			TCCTGTACG/GGAAACA
			GCTATGACCATGATATC
			CTACCGCATGGAAACC
Sequencing Primer set 3	tdTomato insertion	1895 bp	GTAAAACGACGG
			CCAGTCCATCATCAA
			GAACGCCTACA/GG
			AAACAGCTATGACCA
			TGGTGCCTTCA
			CCCAAACTATCT
gRNA	Targeting POMC C-term		CCTACAAGAA
			GGGCGAGTGA
Donor Template	(LHA/P2A/tdTomato/STOP/RHA)		CGACGCTTGACACG
			CCCGACACTGTGCC
			CTGTGTCCTCGGCAC
			GTGGCGAGGGCGGC
			CAGGGCCTAGGCGC
			AGTGACGGGCGCGG
			CAGCCGGGCCGGGG
			TGCGGGGCACGGGC
			TGCCCTCATGCCCTC
			GCGTCTTCCCCCAGG
			AGTGCATCCGGGCCT
			GCAAGCCCGACCTCT
			CGGCCGAGACTCCCA
			TGTTCCCGGGAAATGGC
			GACGAGCAGCCTCTGAC
			CGAGAACCCCCGGAA
			GTACGTCATGGGCCA
			CTTCCGCTGGGACCG
			ATTCGGCCGCCGCAA
			CAGCAGCAGCAGCGG
			CAGCAGCGGCGCAGG
			GCAGAAGCGCGAGGA
			CGTCTCAGCGGGCGA
			AGACTGCGGCCCGCT
			GCCTGAGGGCGGCCC
			CGAGCCCCGCAGCGA
			TGGTGCCAAGCCGGG
			CCCGCGCGAGGGCAA
			GCGCTCCTACTCCATGG
			AGCACTTCCGCTGGGG
			CAAGCCGGTGGGCAAG
			AAGCGGCGCCCAGTGA
			AGGTGTACCCTAACGG
			CGCCGAGGACGAGTCG
			GCCGAGGCCTTCCCCCT
			GGAGTTCAAGAGGGA
			GCTGACTGGCCAGC
			GACTCCGGGAGGGA
			GATGGCCCCGACGG
			CCCTGCCGATGAC
	Antibodies used for immunocytochemistry/flow-cytometry
	Antibody	Dilution	Company Cat #	RRID
			GGCGCAGGGGCCC
			AGGCCGACCTGGA
			GCACAGCCTGCTG
			GTGGCGGCCGAGA
			AGAAGGACGAGGG
			CCCCTACAGGATGG
			AGCACTTCCGCTGG
			GGCAGCCCGCCCAA
			GGACAAGCGCTACG
			GCGGTTTCATGACCT
			CCGAGAAGAGCCAG
			ACGCCCCTGGTGAC
			GCTGTTCAAAAACG
			CCATCATCAAGAAC
			GCCTACAAGAAGGG
			CGAGGGATCCGGAGC
			CACGAACTTCTCTCTG
			TTAAAGCAAGCAGGAG
			ACGTGGAAGAAAACCC
			CGGTCCTATGGTGAGC
			AAGGGCGAGGAGGTCAT
			CAAAGAGTTCATGCGCT
			TCAAGGTGCGCATGGA
			GGGCTCCATGAACGGC
			CACGAGTTCGAGATCG
			AGGGCGAGGGCGAGGG
			CCGCCCCTACGAGGGC
			ACCCAGACCGCCAAGCT
			GAAGGTGACCAAGGGCG
			GCCCCCTGCCCTTCGCC
			TGGGACATCCTGTCCC
			CCCAGTTCATGTACGG
			CTCCAAGGCGTACGTG
			AAGCACCCCGCCGACA
			TCCCCGATTACAAGAAG
			CTGTCCTTCCCCGAGGG
			CTTCAAGTGGGAGCGCG
			TGATGAACTTCGAGGAC
			GGCGGTCTGGTGACC
			GTGACCCAGGACTCC
			TCCCTGCAGGACGG
			CACGCTGATCTACA
			AGGTGAAGATGCG
			CGGCACCAACTTCC
			CCCCCGACGGCCCC
			GTAATGCAGAAGAA
			GACCATGGGCTGGG
			AGGCCTCCACCGAG
			CGCCTGTACCCCCG
			CGACGGCGTGCTGA
			AGGGCGAGATCCAC
			CAGGCCCTGAAGCTG
			AAGGACGGCGGCCAC
			TACCTGGTGGAGTTC
			AAGACCATCTACATG
			GCCAAGAAGCCCGTG
			CAACTGCCCGGCTAC
			TACTACGTGGACACC
			AAGCTGGACATCACC
			TCCCACAACGAGGAC
			TACACCATCGTGGAA
			CAGTACGAGCGCTCC
			GAGGGCCGCCACCAC
			CTGTTCCTGGGGCAT
			GGCACCGGCAGCAC
			CGGCAGCGGCAGCT
			CCGGCACCGCCTCC
			TCCGAGGACAACAA
			CATGGCCGTCATCA
			AAGAGTTCATGCGC
			TTCAAGGTGCGCAT
			GGAGGGCTCCATGAAC
			GGCCACGAGTTCGA
			GATCGAGGGCGAGG
			GCGAGGGCCGCCCC
			TACGAGGGCACCCA
			GACCGCCAAGCTGA
			AGGTGACCAAGGGC
			GGCCCCCTGCCCTT
			CGCCTGGGACATCC
			TGTCCCCCCAGTTC
			ATGTACGGCTCCA
			AGGCGTACGTGAA
			GCACCCCGCCGAC
			ATCCCCGATTACA
			AGAAGCTGTCCTT
			CCCCGAGGGCTT
			CAAGTGGGAGCG
			CGTGATGAACTT
			CGAGGACGGCG
			GTCTGGTGACCG
			TGACCCAGGAC
			TCCTCCCTGCAG
			GACGGCACGCTG
			ATCTACAAGGTGAA
			GATGCGCGGCA
			CCAACTTCCCCCC
			CGACGGCCCCGTA
			ATGCAGAAGAAGA
			CCATGGGCTGGGAGG
			CCTCCACCGAGCGCC
			TGTACCCCCGCGACG
			GCGTGCTGAAGGGCG
			AGATCCACCAGGCCC
			TGAAGCTGAAGGACG
			GCGGCCACTACCTGG
			TGGAGTTCAAGACCA
			TCTACATGGCCAAGA
			AGCCCGTGCAACTGCC
			CGGCTACTACTACGT
			GGACACCAAGCTGGA
			CATCACCTCCCACAA
			CGAGGACTACACCA
			TCGTGGAACAGTAC
			GAGCGCTCCGAGGG
			CCGCCACCACCTGTT
			CCTGTACGGCATGG
			ACGAGCTGTACAAGTGAGGGCACAGCGGGG
			CCCCAGGGCTACCC
			TCCCCCAGGAGGTC
			GACCCCAAAGCCCCT
			TGCTCTCCCCTGCCC
			TGCTGCCGCCTCCCA
			GCCTGGGGGGTCGTGG
			CAGATAATCAGCCTCTT
			AAAGCTGCCTGTAGTTA
			GGAAATAAAACCTTT
			CAAATTTCACATCC
			ACCTCTGACTTTGA
			ATGTAAACTGTGTG
			AATAAAGTAAAAAT
			ACGTAGCCGTCAA
			ATAACAGCAGCA
			TGGATCGGAGGA
			GCACAGTGGTTT
			CCATGCGGTAGG
			ATATTTCACAGG
			ACTTAGTGAGCGT
			GAAAGGAAAATGT
			GCTTCCTGCCCCCA
			CCCCCAAATGGATC
			TTCGAGGGATCAG
			ATAGTTTGGGTGAA
			GGCACAGGGTGGCT
			CCAGCACCTCTAGG
			ATGGCCGTATTTTCC
			ACACACTCCACTGAG
			TGGGAGACTGCTCAG
			CTAGCACACGTGTAA
			AGGCAGGATTCCTG
			CAAGAGTGACCCCGGG
			CGCTCAGGGGCTCC
			CCGGCTCCGGTCCA
			CCTCCAAAAGCCAG
			TTGGACAGGTTGAG
			GCCTACCCACGCCT
			GGGAGTGCTCTGGA
			CTGTCAGGTGAGAA
			GTCTTGGTAATGTCT
			CCGGGGAACTGCTCG
			TAGCAATCCATGGGA
			CCTTAGGCAAGTC
	Primers for top off-target mutagenesis predicted site sequencing (for all CRISP/Cas9, ZNF and TALENS)
	Target	Size of band	Forward/Reverse primer (5′–3′)
Off-target Primer Set 1	Chr5	685	TCCCAAAGTGCTG
			GGATTAC/GATGCT
			GAGGTTTAGGGTACAA
Off-target Primer Set 2	Chr4	274	TGCTATGCCCGGCT
			AATTT/GCCGTCTTG
			GCTCTTCTTTA
Off-target Primer Set 3	Chr13	460	CATTCTGCCTGTCTC
			TCATTCT/CTGTGACCCT
			GACTCTCTATTTG
Off-target Primer Set 4	Chr8	467	CCGGAGTGAATGATGGA
			GATAAG/TGCACACCC
			AAGATCCTTTAG

Among these, clones 1G and 1B2 (homozygous knock-in), and heterozygous clone 10E exhibited typical pluripotent morphology and robust expression of OCT4, SOX2, and NANOG proteins, as confirmed by immunocytochemistry (Fig. 1E and Appendix A). Sanger sequencing verified accurate reporter integration (Fig. 1F and Appendix A), and clone 1G was prioritized for downstream studies due to its homozygous insertion. All reporter lines were karyotyped, and short tandem repeat (STR) profiled (GenePrint 24 System, Promega; see STR analysis), and were free of Mycoplasma contamination (Appendix A).

The pluripotent differentiation potential of the 1G POMC-tdTomato line was validated by directed differentiation into the three germ layers, with expression of lineage markers PAX6 (ectoderm), TBXT (mesoderm), and SOX17 (endoderm) (Fig. 1G). Using our established differentiation protocol (Jovanovic et al., 2024), clone 1G was further directed into hypothalamic arcuate neurons. Live imaging at day 21 of differentiation revealed strong tdTomato fluorescence (Fig. 1H) with an average of 36.57 % of cells expressing tdTomato reporter (95 % confidence interval, margin of error +/− 1.413), and by day 28, td Tomato positive neurons exhibited spontaneous action potentials as demonstrated by patch-clamp electrophysiology recording (Fig. 1I).

Together, the WA09 POMC-tdTomato knock-in reporter line provides a renewable, well-characterized resource for the differentiation, identification, and functional interrogation of POMC-expressing cell types. Knock-in of the P2A sequence at the C-terminus of POMC may alter its stability and interactions leading to haploinsufficiency and/or alterations in functional response, however, this has not been assessed in this study and may require further characterization. This tool will facilitate studies of hypothalamic development, enable disease modeling in metabolic disorders, and allow live cell tracking and selective enrichment of POMC neurons for downstream experimental applications.

3. Materials and methods

3.1. Cell culture and gene editing

Cells were single-cell dissociated with Accutase (Gibco) for 5 min, and 2 × 10⁶ cells were mixed in nucleofection buffer (Cell Nucleofector Kit 2; Cat # VPH-5022) with 7.5 μg plasmid pGS-gRNA POMC (gRNA CCTACAAGAAGGGCGAGTGA), 2.5 Âμg plasmid Cas9_CD4, and 10 μg POMC-P2A-tdTomato targeting vector followed by nucleofection using an Amaxa Nucleofector II (Program A-023) with the Human Stem Cell Nucleofector Kit 2 according to the manufacturer’s instructions. Nucleofected cells were plated onto Geltrex (Gibco)-coated plates in mTeSR Plus (STEMCELL Technologies) with RevitaCell (Gibco). Following 2 days of recovery, CD4-expressing cells were selected using human CD4 MicroBeads (Cat # 130–045-101; MS Column, Cat # 130–042-20; MACS Miltenyi Biotec) and re-plated at clonal density in 10 cm² culture plates. After 7–12 days, colonies were picked into 96-well plates. Clonal analysis was done by PCR for tdTomato, and positive clones were expanded for further analysis. Subsequent culture was done on vitronectin (VTN)-coated plates in Essential 8 medium (E8; Gibco) and cryopreserved using E8 medium with 10 % DMSO and CEPT⁴. All cells were maintained at 37 °C, 5 % CO₂ and assayed at passage 32.

3.2. Directed differentiation

Ectoderm, mesoderm and endoderm were induced as previously described (Chen et al., 2021).

Cell lines were differentiated into hypothalamic arcuate neurons as previously described (Jovanovic, et al., 2024). Briefly, WA09 POMC-tdTomato cells were plated on VTN-coated plates in E8. The next day, medium was replaced with Arc-1. On day 7, cells were single cell dissociated and plated onto AggreWell plates. The cells were maintained in Arc-2 medium until day 11. On day 11, medium was replaced with Arc-3. Spheres were dissociated on day 14 and plated on Geltrex-coated plates in Arc-3 medium until day 28, when POMC-tdTomato expression was assessed.

3.3. Immunocytochemistry

POMC-tdTomato cells were fixed with 4 % paraformaldehyde and stained in Perm-block buffer (0.1 % Triton X-100 and 5 % Bovine Serum Albumin in DPBS) using primary antibodies overnight and secondary antibodies for 2 h at RT (Table 2). Nuclei were stained using Hoechst 33,342 (ThermoFisher). Images were captured using a Leica DMi8 epifluorescence microscope.

3.4. Mycoplasma detection

The MycoAlert PLUS Mycoplasma Detection Kit (Lonza) was used to verify that WA09 POMC-tdTomato cell lines were mycoplasma-free.

3.5. tdTomato knock-in validation

Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen). PCR reactions were performed using appropriate primers (Table 2). PCR products were Sanger sequenced.

3.6. Short tandem repeat (STR) and karyotype analysis

STR profiling and karyotyping were performed by Cell Line Genetics (Madison, WI).

3.7. Patch-clamp electrophysiology

POMC-tdTomato (1G clone)-expressing hypothalamic neurons were plated on 12 mm glass coverslips (Carolina Biological Supply) coated with Poly-l-Ornithine (Sigma Aldrich) and iMatrix-511 SILK (Amsbio). D28-D35 tdTomato-expressing Arc neurons were visually identified by fluorescence for patch-clamp electrophysiology recordings. Extracellular media contained BrainPhys media (STEMCELL Technologies). Intracellular solution contained (in mM): 135 K-gluconate, 5 KCl, 5 MgATP, 0.5 Na2GTP, 5 HEPES, 2 MgCl2, 5 EGTA, 0.5 CaCl2, pH 7.4. Recordings were made using an Axopatch 200B (Molecular Devices) amplifier at a sampling rate of 10 kHz and a 2 kHz Bessel low-pass filter. Data was digitized using a Digidata 1550B with pClamp11 (Molecular Devices). Whole-cell configuration was obtained in voltage clamp mode, and switched to current clamp mode after waiting 3 min for the membrane potential to stabilize. Spontaneous action potentials were recorded at a − 50 mV.

Resource Table:

Unique stem cell line identifier	WAe009-A-3I WAe009-A-3 J WAe009-A-3 K
Alternative name(s) of stem cell line	WA09 POMC-tdTomato (homozygous clone 1G) WA09 POMC-tdTomato (homozygous clone 1B2) WA09 POMC-tdTomato (heterozygous clone 10E)
Institution	National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA Department of Pathology and Cell Biology, Columbia University, New York, NY, USA
Contact information of the reported cell line distributor	Carlos Tristan carlos.tristan@nih.gov
Type of cell line	ESC
Origin	Human embryonic stem cell line (WA09; H9)
Additional origin info (applicable for human ESC or iPSC)	Age: Sex: Female Ethnicity: Unknown
Cell Source	WA09 (H9) (WiCell Research Institute, Inc.)
Method of reprogramming	Not applicable
Clonality	Clonal
Evidence of the reprogramming transgene loss (including genomic copy if applicable)	Not applicable
The cell culture system used	Essential 8 medium on vitronectin
Type of the Genetic Modification	CRISPR Cas9 mediated insertion of tdTomato at the POMC locus.
Associated disease	N/A
Gene/locus modified in the reported transgenic line	POMC
Method of modification / user-customizable nucleases (UCN) used, the resource used for design optimization	CRISPR Cas9
User-customizable nuclease (UCN) delivery method	Nucleofection
All double-stranded DNA genetic material molecules introduced into the cells	Not applicable
Evidence of the absence of random integration of any plasmids or DS DNA introduced into the cells.	Not applicable
Analysis of the nuclease-targeted allele status	Sanger sequencing of target region.
Homozygous allele status validation	PCR spanning the editing site, followed by Sanger sequencing.
Method of the off-target nuclease activity prediction and surveillance	Cas-OFFinder with 3 mismatches. PCR across four off-target sites.
Descriptive name of the transgene	POMC-tdTomato
Eukaryotic selective agent resistance cassettes (including inducible, gene/cell type-specific)	Not applicable
Inducible/constitutive expression system details	Not applicable
Date archived/stock creation date	4/24/25
Cell line repository/bank	Not applicable
Ethical/GMO work approvals	Not applicable
Addgene/public access repository recombinant DNA sources’ disclaimers (if applicable)	Not applicable

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scr.2026.103905.

Data availability

Data will be made available on request.