Establishment and characterization of CSCRi006-A: an induced pluripotent stem cell line generated from a patient with Diamond-Blackfan Anemia (DBA) carrying ribosomal protein S19 (RPS19) mutation

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia characterized by ineffective erythropoiesis. DBA is majorly caused by mutations in the ribosomal protein (RP) genes (Gadhiya and Wills in Diamond-Blackfan Anemia, https://www.statpearls.com/; 2023). A suitable disease model that yields a continuous supply of erythroid cells is required to study disease pathogenesis and drug discovery. Toward this, we reprogrammed dermal fibroblasts from a DBA patient with a heterozygous mutation c.22-23delAG in the RPS19 gene identified through exome sequencing. To generate induced pluripotent stem cells (iPSCs), we induced episomal expression of the reprogramming factors OTC3/4, L-MYC, LIN28, SOX2, and KLF4, and a p53 shRNA2. The DBA-iPSC line CSCRi006-A generated during this study was extensively characterized for its pluripotency and genome stability. The clone retained normal karyotype and showed high expression levels of pluripotency markers, OCT4, NANOG, SOX2, TRA-I-60, TRA-I-81, and SSEA4. It could differentiate into cells originating from all three germ cell layers, as identified by immunostaining for SOX17 (endoderm), Brachyury (mesoderm), and PAX6 (ectoderm). IPSCs provide a renewable source of cells for in vitro disease modeling. CSCRi006-A, a thoroughly characterized iPSC line carrying heterozygous RPS19 c.22-23delAG mutation, is a valuable cell line for the disease modeling of DBA. This iPSC line can be differentiated into different blood cell types to study the mechanisms of disease development and identify potential treatments.

Although RPs are critical for protein synthesis in all cell types, their haploinsufficiency selectively perturbs erythropoiesis, causing relatively less impact on the development of other blood cell lineages.This observation highlights the crucial role of ribosome biogenesis in erythroid differentiation.Understanding the pathogenesis of DBA and the role of RPs in normal erythropoiesis is essential for improving treatment options and gaining valuable insights into the broader cellular and molecular mechanisms that govern normal erythropoiesis.Frequent blood sampling from DBA patients for extensive ex-vivo erythropoiesis experiments is technically challenging due to severe anemia in these patients, necessitating regular blood transfusions.
An alternative approach for studying molecular dysfunctions in DBA is the use of patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells.These iPSCs offer an unlimited cell source for disease modeling and the study of molecular dysfunctions [6][7][8].By differentiating patient-specific iPSCs into affected target cell types, such as hematopoietic cells, researchers have successfully modeled various hematological diseases [8,9].DBA exhibits heterogeneity in genotypes and phenotypes but converges into a typical erythroid hypoplasia phenotype in patients.Therefore, disease models with different mutations are valuable for investigating the molecular basis of erythroid perturbation in DBA and expanding our understanding of erythroid development.In this study, we generated iPSCs from a DBA patient with a heterozygous mutation c.22-23delAG in the RPS19 gene using non-integrating episomal expression of the reprogramming factors, OTC3/4, L-MYC, LIN28, SOX2 and KLF4, and a p53 shRNA [2].

Derivation of fibroblasts
Dermal fibroblasts were isolated and cultured using a previously described protocol [10].In brief, 1 mm 3 skin biopsies obtained from the patients were washed with phosphatebuffered saline (PBS) (HyClone) and incubated overnight at 37 °C/5% CO 2 in a 15 mL conical flask with 1 mL of a skin biopsy digestion medium, containing DMEM with 20% FBS, 0.25% collagenase type I (Thermo Fisher Scientific), 0.05% DNase I (Merck, and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin).The tube was briefly vortexed the next day, and 7 mL of DMEM containing 20% FBS was added.The dissociated cells were then plated in a T25 culture flask and incubated for 3 days at 37 °C/5% CO 2 .The medium was replaced on the fourth day with DMEM containing 10% FBS and antibiotics.The cells were cultured with a media change on alternate days until they reached approximately 80% confluence.Cells were then passaged at a 1:4 ratio using 0.05% trypsin-EDTA (Thermo Fisher Scientific).After two passages, the cells were cryopreserved.

Next-generation sequencing
DNA extracted from the blood sample was subjected to targeted exome sequencing using a custom capture kit for 6763 genes documented in OMIM [11].The libraries were sequenced to mean > 80-100 × coverage on the Illumina sequencing platform.The sequence reads were aligned to the human reference genome (GRCh37/hg19) using BWA program [12,13] and analyzed using Picard and GATK version 3.6 to identify variants relevant to the clinical indication [14].Gene annotation of the variants was performed using the VP program against the Ensembl release 87 human gene model [15].Clinically relevant mutations were annotated using published variants in the literature and a set of disease databases-ClinVar, OMIM, GWAS, HGMD, and SwissVar [11,16,17].Common variants were re-filtered based on allele frequency in 1000 Genome Phase 3, ExAC, EVS, dbNP147, 1000 Japanese Genome, and Indian population database [18,19].Non-synonymous missense, null, and splice site junction variants were considered for identifying the disease-associated mutations.The effect of nonsynonymous missense variants was analyzed using multiple algorithms, such as PolyPhen-2, SIFT, MutationTaster2, Mutation Assessor, and LRT.

Immunofluorescence analysis
To assess pluripotency, iPSC colonies were fixed by treating with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% TritonX-100 (Sigma) in phosphate buffer saline (PBS) for 15 min and blocking with 1% bovine serum albumin (BSA) and 5% fetal bovine serum (FBS) in PBS for 20 min at room temperature.Subsequently, the cells were incubated with primary antibodies (Table 1) in the blocking buffer overnight (16 h) at 4 °C, followed by incubation with suitable labeled secondary antibodies for 2 h at room temperature.For nuclear staining, the cells were incubated with 4',6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature.The staining was visualized under a fluorescence microscope (DS6000, Leica Microsystems) with appropriate filters.

Real-time PCR analysis
RNA was extracted from iPSC clones using NucleoZOL (Takara Bio) following the manufacturer's protocol.1 μg of total RNA was used for reverse transcription using Prime-Script™ RT Reagent (Takara Bio) as per the manufacturer's protocol.Quantitative real-time polymerase chain reaction (RT-PCR) was set up with TB Green Premix Ex Taq II (Tli RNase H Plus) (Takara) using specific primers and analyzed with QuantStudio12K Flex (Life Technologies) real-time PCR systems.The list of primers is given in Table 2.

Trilineage differentiation
To induce trilineage differentiation, iPSCs were dissociated into single cells using TrypLE™ Select (Gibco) and plated in mTeSR Plus medium (StemCell Technologies) containing 10 μM revitacell (Thermo Fisher Scientific).On day 1, an appropriate volume of pre-warmed (37 °C) STEMdiff™ Trilineage medium for ectoderm, endoderm, and mesoderm differentiation was added to each well, and the plates were incubated at 37 °C for 24 h.Medium change was done until day 5 (mesoderm and endoderm lineages) or day 7 (ectoderm lineage).Immunofluorescence was performed as per the method described above.

Karyotyping
Karyotyping was performed using a previously described method [20].Briefly, the iPSC colonies in culture were exposed to 200 μg/ml colcemid for 30 min and harvested as a single-cell suspension with 0.05% trypsin.The cell pellet was treated with 0.075 M KCl solution for 12 min at 37 °C and then fixed using modified Carnoy's fixative (methanol and acetic acid in the ratio 3:1), followed by centrifugation at 1000 rpm for 10 min at room temperature.The fixed cells resuspended in 5 ml of modified Carnoy's fixative were spread on a slide and stained according to the standard cytogenetics protocols.Images were acquired using the AxioImager A1 (Carl Zeiss, Germany) and analyzed using Ikaros Software (Metasystems, Germany).

Short tandem repeats (STR) analysis and Sanger sequencing.
Genomic DNA was isolated from the DBA-iPSC, a normal iPSC line (N-iPSC), and the patient's peripheral blood using the Gentra Purgene DNA isolation kit (Qiagen).Short tandem repeats (STRs) were analyzed using PowerPlex ESI 17 Fast Systems (Promega) following the manufacturer's protocol, and fragment length analysis was performed on an ABI 3130 Genetic Analyzer (Thermo Fisher Scientific) using Genemapper v4 software (Thermo Fisher Scientific).To confirm the RPS19 mutation identified by whole exome sequencing, primers were designed to amplify exon 2 of RPS19, and the forward primer was used for Sanger sequencing (Table 2).

Integration PCR
Genomic DNA was isolated as described above.PCRs were performed with primers (Table 2) designed to amplify the transgenes.The amplified PCR products were analyzed on 1%-agarose gel. 5 ng of plasmids were used as positive controls.A previously characterized integration-free iPSC clone generated from a healthy donor was used as a negative control [21].

Mycoplasma detection assay
Mycoplasma detection was performed using MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza) following the manufacturer's protocol.Briefly, 2 mL of cell culture supernatant was centrifuged at 200×g for 5 min to pellet any cells.Then, 100 µL of the supernatant was transferred into one well of a 96-well plate.Next, 100 µL of MycoAlert™ PLUS Reagent was added to each sample and incubated for 5 min.Luminescence was measured by SpectraMax i3X (Molecular Devices) using the software SoftMax Pro 7 and recorded as reading A. Then, 100 µL of MycoAlert™ PLUS Substrate was added to each sample and incubated for 10 min.Luminescence was measured and recorded as reading B. The ratio was calculated as reading B/reading A. A ratio > 1.2 indicates mycoplasma contamination, while a ratio < 1 indicates the absence of mycoplasma in the cell culture.

Hematopoietic differentiation
The iPSCs were subjected to hematopoietic differentiation using STEMdiff hematopoietic kit (Stem Cell Technologies) following the manufacturer's protocol.Initially, iPSCs were dissociated into small clumps of ~ 100-200 µm size and 80-100 colonies were seeded per well of a 12-well plate coated with GFR-Matrigel (Corning) containing mTeSR Plus medium (Stem Cell Technologies).On day 0 of differentiation, mTeSR Plus medium was replaced with 1 mL of STEMdiff medium A (SDA).On day 2, a half-medium change was performed using SDA.On day 3, the mesoderm cells derived from iPSCs in the SDA medium were dissociated into single cells using TryplE-Express (Thermofisher Scientific), and 6X10 4 cells were seeded again in SDA per well of a 24-well plate in triplicate, without any cell attachment matrix.On day 4, SDA was replaced with SDB medium.Half medium change with SDB was done every alternate day until day 12 of the culture.

Mutation analysis
A heterozygous 2 base pair deletion in exon 2 of the RPS19 gene (chr19:12364865_42364866delAG) was identified in the patient's peripheral blood DNA.This mutation leads to a frameshift and premature truncation of the protein, resulting in a termination codon at the amino acid position 42 (p.Asp8Arg(sTer42; ENST00000598742).This RPS19 variant has not been reported in the 1200 genomes ExAC and the Indian human genome databases.No additional mutations associated with congenital anemia were identified in any other genes in the 6763 genes analyzed in the targeted exome sequencing panel.To validate the finding, we performed Sanger sequencing for the patient's fibroblasts and blood samples, as well as for the iPSC line, CSCRi006-A, derived from the patient's fibroblasts.The mutation was confirmed in the fibroblasts and the patient's peripheral blood cells as well as in the DBA-iPSC (Fig. 1b).However, this mutation was not detected in the peripheral blood cells of the patient's parents.

Generation of iPSCs
The dermal fibroblasts used for reprogramming were obtained from a skin biopsy obtained with informed consent approved by the institutional science and ethics review board of Christian Medical College, Vellore, India.Patient and control adult dermal fibroblasts at an early passage (passage 4) were reprogrammed using episomal expression of reprogramming genes, OTC3/4, L-MYC, LIN28, SOX2, and KLF4, and a p53 shRNA [22].The early passage was chosen for reprogramming as the senescence of fibroblasts in later passages reduces reprogramming efficiency [23] (Fig. 2a).We observed that reprogramming efficiency was significantly lower in DBA fibroblasts compared to normal control fibroblasts.The extremely low reprogramming efficiency from DBA fibroblasts has been reported earlier [23].

Characterization of iPSCs
One of the iPSC lines, derived from the DBA patient's fibroblasts, CSCRi006-A, was extensively characterized for its genome stability and pluripotency.This iPSC clone exhibited typical morphology of iPSCs (Fig. 2c, d), exhibited high expression levels of pluripotency markers (Fig. 3), and had a normal karyotype (Fig. 4).An iPSC line derived from a normal individual (N-iPSC) in our laboratory was used as a control for pluripotency analysis [1].Sanger sequencing confirmed the presence of the patient's mutation in the DBA-iPSC clone (Fig. 1b).Analysis of 17 short tandem repeats in the DBA-iPSC clone showed an identical pattern to that obtained with the patient's blood cells (Fig. S1).
Integration PCR confirmed the elimination of reprogramming vectors from the iPSC clone.For this assay, a 1031 bp genomic fragment amplified from the β-globin cluster was used as a positive control for checking the DNA integrity (Fig. 1a).The pluripotency of the DBA-iPSC was further confirmed by its ability to differentiate into three primary germ layers, as evidenced by the expression of PAX6 for ectoderm, SOX17 for endoderm, and Brachyury for mesoderm (Fig. 5).Notably, hematopoietic differentiation of the DBA-iPSC line was significantly impaired compared to the N-iPSC line (Fig. 6).The number of hematopoietic stem and progenitor cells (HSPCs) derived from the DBA-iPSC was significantly lower, indicating a deficiency in their ability to generate these crucial cell types.However, it is noteworthy that despite the defective hematopoietic differentiation observed in the DBA-iPSC-derived HSPCs, the expression levels of CD34 and CD45, two cell surface markers commonly used for detecting hematopoietic cells derived from iPSCs, were found to be similar to those expressed in the HSPCs derived from N-iPSC (Fig. S2).

Discussion
iPSCs offer a renewable and abundant source of cells for disease modeling, enabling the research on studying human diseases at the cellular level.DBA is a rare genetic disorder characterized by the inability of the bone marrow to produce red blood cells, resulting in severe anemia.Investigating DBA pathogenesis using primary cells from patients has been challenging due to the limited supply of patients' blood cells.The iPSCs generated from DBA patients provide a continuous supply of blood cells as an alternative to primary cells for disease modeling.
In this context, we successfully generated an iPSC line, CSCRi006-A, from a DBA patient with a heterozygous mutation in the RPS19 gene.Extensive characterization of this DBA-iPSC line was conducted to ensure its quality and suitability for downstream applications.It was free from reprogramming vector integration and showed normal karyotype, essential features for ensuring that the iPSC line is stable and retains its properties during long-term culture.Furthermore, the iPSC line was able to differentiate into cells from all three embryonic germ layers, ectoderm, mesoderm, and endoderm, indicating the potential of the iPSC line to differentiate into various cell types, including those that exhibit the disease phenotypes, such as erythroid cells.The successful generation of this DBA-iPSC line provides a valuable tool for understanding the disease mechanisms and developing novel therapeutic approaches.Our findings from hematopoietic differentiation revealed a specific and severe impairment

Fig. 2 Fig. 3
Fig. 2 Establishment of the DBA-iPSC line, CSCRi006-A, from dermal fibroblasts.a Fibroblasts at passage 4 (100X magnification).b Morphology of a DBA-iPSC clone emerging from the reprogramming fibroblasts.c, d Morphology of CSCRi006-A iPSC at passage 15 at 100X and at 200X magnifications.respectively

Fig. 6
Fig. 6 Hematopoietic differentiation of the DBA-iPSC and normal iPSC (N-iPSC) line.a, b Phase-contrast images of HSPCs generated on the differentiation day 12 from a normal iPSC line (a) and DBA-

Table 1
Antibodies used for immunofluorescence (IF) and flow cytometry

Table 2
The