Generation of a human induced pluripotent stem cell line from a patient diagnosed with schizophrenia carrying a 16p11.2 deletion

Abstract

16p11.2 copy number variations have been associated with neurodevelopmental disorders. Human induced pluripotent stem cells were generated from fibroblasts obtained from a patient diagnosed with schizophrenia with a 16p11.2 deletion. The generated cell line was further validated for its pluripotency and potential to differentiate into the three germ layers.

1. Resource table

Unique stem cell line identifier	UIi001-A https://hpscreg.eu/cell-line/UIi001-A
Alternative name(s) of stem cell line	SCZ019
Institution	University of Iowa Carver College of Medicine, Iowa Neuroscience Institute
Contact information of distributor	Aislinn Williams, aislinn-williams@uiowa.edu
Type of cell line	iPSC
Origin	Human
Additional origin info required for human ESC or iPSC	Age: 33
	Sex: M
	Ethnicity if known: White, not Hispanic
Cell Source	Fibroblasts
Clonality	Clonal
Associated disease	Schizophrenia
Gene/locus	16p11.2 deletion (548 kb, Chr16: 29558172–30106541)
Date archived/stock date	March 2019
Cell line repository/bank	N/A
Ethical approval	University of Iowa Institutional Review Board approval number 201805995 University of Iowa Human Pluripotent Stem Cell Committee approval number 2017–02

2. Resource utility

Copy number variations (CNV) of 16p11.2 are implicated in neuro-developmental disorders such as autism spectrum disorders (ASD) and schizophrenia. This iPSC line was generated from a patient diagnosed with schizophrenia with a 16p11.2 deletion and can be used for in vitro studies investigating the developmental aspects of neuropsychiatric disorders.

3. Resource details

Copy number variation in the 16p11.2 locus, both duplications and deletions, are associated with neurodevelopmental disorders. This region contains approximately 29 genes, including BCKDK, CORO1A, KCTD13, MAPK3, SETD1A, SEZ6L2, SRCAP, and TAOK2. Deletions in the 16p11.2 locus are most commonly associated with autism spectrum disorders, intellectual disability, and developmental delay. 16p11.2 deletion syndrome is associated with multiple variably penetrant phenotypes, including speech articulation abnormalities, limb and trunk hypotonia, hyporeflexia, seizures, macrocephaly, and Chiari I malformation (Steinman et al., 2016). Although 16p11.2 duplication has most frequently been associated with psychotic symptoms whereas the deletion is most commonly associated with ASD, studies have also shown 16p11.2 deletion carriers can present with psychotic symptoms and have been diagnosed with schizophrenia (Jutla et al., 2020; Niarchou et al., 2019). Thus, the iPSC line generated from a patient diagnosed with schizophrenia carrying the 16p11.2 deletion is a unique resource for investigating the cellular mechanisms of the overlapping phenotypes observed in patients with autism and psychosis in 16p11.2 deletion syndromes (Table 1).

Table 1.

Characterization and validation.

Classification	Test	Result	Data
Morphology	Photography Bright field	Normal	Fig. 1 panel A
Phenotype	Qualitative analysis Immunohistochemistry and RT-PCR	Positive staining for Nanog, Oct3/4, Sox-2, and Tra-1–60 and positive for pluripotency gene expression; NANOG, OCT3/4, SOX-2	Fig. 1 panel D and E
	Quantitative analysis	OCT3/4: 99% NANOG: 93%	Fig. 1 panel D, Supplementary figure panel A
Genotype	Karyotype (G-banding) and resolution	46XY, Resolution 400–475	Fig. 1 panel B
Identity	Microsatellite PCR (mPCR) OR	not performed	N/A
	STR analysis	16 sites tested are tested, 100% match	Submitted in archive with journal
Mutation analysis (IF APPLICABLE)	Sequencing	N/A	N/A
	Southern Blot OR WGS	N/A	N/A
Microbiology and virology	Mycoplasma	Mycoplasma testing by PCR. Negative	Supplementary figure panel B
Differentiation potential	Embryoid body formation	Positive for the three germ layers. mesoderm: Brachyury, VE-Cadherin; ectoderm: Tuj1, Krt-18; endoderm AFP, GATA-4	Fig. 1 panel F and G
List of recommended germ layer markers	Expression of these markers has to be demonstrated at mRNA (RT PCR) or protein (IF) levels, at least 2 markers need to be shown per germ layer	Ectoderm: PAX6, SOX1, OTX1/2, TUBB3/TUJ1, MAP2, NeuN (at least 1 marker from the set above, also acceptable 1 sub-lineage specific cell type marker, e.g. DCX, SYN1, TBR2); Endoderm: SOX17, FOXA2, LEFTY1 (at least 1 marker from the set above, also acceptable 1 sub-lineage specific cell type marker, e.g., AFP, PDX1) Mesoderm: BRACHYURY/TBXT, TBX6, HAND1 (at least 1 marker from the set above, also acceptable 1 sub-lineage specific cell type marker, e.g. A-SMA, PAX3/7, NKX2.5)	RT-PCR with reference genes: mesoderm: Brachyury, VE-Cadherin; ectoderm: Tuj1, Krt-18; endoderm AFP, GATA-4
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
	HLA tissue typing	N/A	N/A

Fibroblasts were obtained from a 33-year old white male diagnosed with schizophrenia (Flaum et al., 1992) with a 16p11.2 deletion (548 kb, Chr16: 29558172–30106541, hg18 assembly) CNV. iPSCs were generated from these fibroblasts using an episomal vector kit. Colonies were selected and expanded (Fig. 1A). Loss of episomal vectors from the iPSCs was validated by PCR from cell DNA (Fig. 1C). iPSCs were tested for Mycoplasma which was not detected (Supplementary figure B). iPSCs were further analyzed for their karyotype using the G-banding method and no abnormality was observed (Fig. 1B). Short tandem repeat (STR) analysis confirmed that the iPSCs and the fibroblasts were from the same donor. The pluripotency of the iPSCs was checked by immunolabeling against Nanog, Oct3/4, Sox2, and Tra-1–60 (Fig. 1D, Supplementary Figure A, Table 2) and RT-PCR to verify mRNA expression of Nanog, Oct3/4, and Sox2 (Fig. 1E). Both on protein and mRNA levels, cells were positive for pluripotency markers. Moreover, differentiation potential into the three germ layers in vitro was tested by spontaneous embryoid body (EB) formation followed by RT-PCR. iPSC colonies formed healthy EBs (Fig. 1F) which expressed mesoderm (Brachyury, VE-Cadherin), ectoderm (Tuj1, Krt-18), and endoderm (AFP, GATA-4) layer cell markers (Fig. 1G).

Fig. 1.

Characterization and quality control data for iPSC line Uii001-A.

Table 2.

Reagents details.

	Antibodies used for immunocytochemistry/flow-cytometry
	Antibody	Dilution	Company Cat #	RRID
Pluripotency markers	Rabbit anti Nanog	1:200	Abcam Cat# ab21624	RRID: AB_446437
	Mouse anti Oct3/4	1:200	Santa Cruz Biotechnology Cat# sc-5279	RRID: AB_628051
	Rabbit anti Sox-2	1:800	Millipore Cat# AB5603	RRID: AB_2286686
	Mouse anti Tra-1–60	1:200	Millipore Cat# MAB4360	RRID: AB_2119183
Secondary antibodies	Donkey anti-rabbit IgG (H + L) Alexa Fluor 488	1:200	Jackson ImmunoResearch Labs Cat# 711–545-152	RRID:AB_2313584
	Donkey anti-mouse IgG (H + L) Alexa Fluor 594	1:200	Jackson ImmunoResearch Labs Cat# 715–585-151	RRID:AB_2310855
	Donkey anti-rabbit IgG (H + L) Alexa Fluor 594	1:800	Jackson ImmunoResearch Labs Cat# 711–585-152	RRID: AB_2340621
	Donkey anti-mouse IgG (H + L) Alexa Fluor 488	1:200	Jackson ImmunoResearch Labs Cat# 715–545-151	RRID:AB_2341099
	Primers
	Target	Size of band	Forward/Reverse primer (5′−3′)
Episomal vectors	OriP	544 bp	Forward: TTCCACGAGGGTAGTGAACC
			Reverse: TCGGGGGTGTTAGAGACAAC
	EBNA1	666 bp	Forward: ATCGTCAAAGCTGCACACAG
			Reverse: CCCAGGAGTCCCAGTAGTCA
Pluripotency Markers (qPCR)	NANOG	112 bp	Forward: TCCTCCTCTTCCTCTATACTAAC
			Reverse: CCCACAAATCACAGGCATAG
	OCT3/4	131 bp	Forward: AGTCAGTGAACAGGGAATGG
			Reverse: TCGGGATTCAAGAACCTACG
	SOX-2	140 bp	Forward: GAGAGAAAGAAAGGGAGAGAAG
			Reverse: GAGAGAGGCAAACTGGAATC
Differentiation markers	Brachyury	165 bp	Forward: ACCCAGTTCATAGCGGTGAC
			Reverse: GGATTGGGAGTACCCAGGTT
	VE-Cadherin	229 bp	Forward: CCTACCAGCCCAAAGTGTGT
			Reverse: GAGATGACCACGGGTAGGAA
	TUJ1	237 bp	Forward: ATGCGGGAGATCGTGCACAT
			Reverse: CCCTGAGCGGACACTGT
	Krt-18	164 bp	Forward: CACAGTCTGCTGAGGTTGGA
			Reverse: GAGCTGCTCCATCTGTAGGG
	AFP	94 bp	Forward: AAACTATTGGCCTGTGGCGA
			Reverse: GGCCAACACCAGGGTTTACT
	GATA-4	184 bp	Forward: CAGATGCCTTTACACGCTGA
			Reverse: TCCGCTTGTTCTCAGATCCT
House-Keeping Genes (qPCR)	Beta-ACTIN	143 bp	Forward: GCCGAGGACTTTGATTGC
			Reverse: GTGTGGACTTGGGAGAGG
Mycoplasma	5′ Myc#1	~500 bp	CGC CTG AGT AGT ACG TTC GC
	5′ Myc#2		CGC CTG AGT AGT ACG TAC GC
	5′ Myc#3		TGC CTG AGT AGT ACA TTC GC
	5′ Myc#4		CGC CTG GGT AGT ACA TTC GC
	5′ Myc#5		CGC CTG AGT AGT ATG CTC GC
	5′ Myc#6		TGC CTG GGT AGT ACA TTC GC
	3′ Myc#1		GCG GTG TGT ACA AGA CCC GA
	3′ Myc#2		GCG GTG TGT ACA AAA CCC GA
	3′ Myc#3		GCG GTG TGT ACA AAC CCC GA

4. Materials and methods

4.1. Fibroblast collection and reprogramming

Skin biopsy was obtained from the patient after obtaining informed consent. Fibroblasts were cultured in DMEM (Gibco) with 15% FBS (Atlanta Biologicals) and 1% MEM-NEAA (Gibco) at 37 °C and 5% CO₂. Fibroblasts were reprogrammed using the Epi5 Episomal iPSC reprogramming kit (LifeTechnologies) following the manufacturer’s protocol using the 4D Nucleofector core unit (Lonza) for electroporation.

4.2. Cell culture

iPSCs were maintained in TeSR-E8 (Stemcell Technologies) on Matrigel (0.08 mg/ml) coated tissue culture plates at 37 °C and 5% CO₂. Cells were passaged 1:2–1:3 using 1 mM sodium citrate passaging solution every 3–4 days.

4.3. Loss of episomal vector

To confirm the loss of episomal vectors in the iPSCs, DNA was isolated from reprogrammed cells at p22 with the NucleoSpin Tissue kit (Macherey-Nagel) following the manufacturer’s protocol. DNA was tested for episomal vector sequences by PCR using GoTaq Green Master Mix (Promega), including fibroblast DNA as a negative control and the vector mix as a positive control. The PCR cycle was: 2 min 94 °C, 35 × (30 s 94 °C, 30 s 65 °C, 1 min 72 °C), 7 min 72 °C, ∞ 4 °C.

4.4. Pluripotency

iPSCs at p6 were fixed with 4% PFA for 10 min and washed with DPBS. Fixed cells were incubated in primary antibodies overnight at 4 °C and in secondary antibodies for 30 min at room temperature. Nuclear staining was done with DAPI. Images were acquired using an Olympus IX83 epifluorescence microscope. Random areas on the DAPI images were selected for cell counting. Cells positive for pluripotency markers (Nanog or Oct3/4) were manually counted using ImageJ Software and reported as the percentage of total cells (DAPI).

RNA was isolated from cells at p10 using TRIzol (Invitrogen) following the manufacturer’s protocol. RT-PCR for pluripotency markers was performed by generating cDNA according to the manufacturer’s protocol (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems). PCR was performed using GoTaq Green Master Mix (Promega) and samples were run on 1% agarose gel. The PCR cycle was: 5 min 95 °C, 38 × (15 s 95 °C, 30 s 55 °C, 45 s 72 °C), 5 min 72 °C, ∞ 4 °C.

4.5. Embryoid body formation

Embryoid bodies (EB) were generated by treating iPSC colonies with 1 mM sodium citrate passaging solution, then manually lifting colonies in hPSC culture medium (DMEM/F12, 10% KOSR, 0.5% GlutaMax/B-mercaptoethanol, 1% NEAA). EBs were collected on day 10 and lysed in TRIzol reagent. RT-PCR for the three germ layers was done with the generated cDNA. The PCR cycle was: 5 min 95 °C, 38 × (15 s 95 °C, 30 s 55 °C, 45 s 72 °C), 5 min 72 °C, ∞ 4 °C.

4.6. CNV identification

The CNV was detected by array comparative genomic hybridization (aCGH) following the manufacturer’s protocol (Roche NimbleGen cgh_cnv_userguide_v7p0). Briefly, case and control DNA (from an unaffected Filipino male) were labeled with Cy3- and Cy5-coupled non-amers respectively. Each labeled DNA was cohybridized to a Roche NimbleGen human whole-genome tiling microarray (Human CGH 2.1 M Whole-Genome Tiling v2.0D Array). The array was washed, scanned, and analyzed.

4.7. Karyotyping and STR

Cells were grown in vitro and arrested at metaphase with colcemid at p14. Chromosomes were stained by the G-banding method. The chromosome number was determined by microscopic analysis and cells were examined for the presence or absence of detectable structural rearrangements. Karyotypes were prepared from computer assisted digital images of these metaphases.

To confirm the donor identity of iPSCs, we isolated DNA from iPSCs and the parent fibroblasts using the NucleoSpin Tissue kit (Macherey-Nagel) following the manufacturer’s protocol and submitted the samples to University of Arizona Genetics Core for short tandem repeat (STR) analysis.

4.8. Mycoplasma test

The presence of mycoplasma was tested using a PCR assay (Uphoff and Drexler, 2002). Spent medium was collected at p11 and centrifuged. Supernatant was discarded and pellet was resuspended in buffer containing 50 mM Tris-HCl, 100 mM EDTA, 100 mM NaCl, 1% SDS, pH 8.0, and incubated at 95 °C for 15 min. Protein was pelleted using 7.5 M ammonium acetate on ice for 5 min, followed by centrifugation at 16,000 × g for 10 min. DNA was precipitated from the supernatant using isopropanol, dried, and then rehydrated in water for PCR testing. An internal control DNA was added to each sample (except for the negative control where only water was included as a template) as a control for PCR integrity. For the positive control, we used mycoplasma DNA as a template. The mycoplasma DNA positive control template and the internal control template were both obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). GoTaq Green Master Mix (Promega) was used for the PCR and the cycle was: 2 min 94 °C, 39 × (30 s 94 °C, 30 s 65 °C, 30 s 72 °C), 2 min 72 °C, ∞ 12 °C.