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Development of pathophysiologically relevant models of sickle cell disease and β-thalassemia for therapeutic studies – Nature Communications

CD34+ Hematopoietic stem progenitor cell (HSPCs) culture

Peripheral blood from healthy donors, BT, and SCD patients eligible for research purposes were obtained under written informed consent. The samples obtained from human donors were de-identified and no information was available on the sex of the donors. The study was approved by the Institutional Human Ethics Committees at Institute of Genomics and Integrative Biology and Thalassemia and sickle cell society (CSIR-IGIB/IHEC/2017-18/12; TSCS-1112018; 2020-001-EMP-28) and used according to the Declaration of Helsinki. The blood samples were subjected to peripheral blood mononuclear cells (PBMC) isolation by the Ficoll gradient method50 followed by CD34+ cells purification. The purification was performed using EasySep™ Human CD34 positive selection kit II (Stem cell Technologies) with the help of EasySep™ Magnet (STEMCELL Technologies) as per the manufacturer’s instructions. The purified CD34+ HSPCs were cultured in StemSpan™ SFEM II medium supplemented with CD34+ Expansion cytokines (STEMCELL Technologies) for maintenance and expansion. The cells were cultured for 6 days followed by erythroid differentiation.

sgRNA design and validation

Single guide RNAs (sgRNAs) were designed using CRISPR/cas9 guide RNA design checker (IDT) and CHOPCHOP51. The most efficient sgRNAs with high specificity and the least off-target effects were chosen. All the sgRNAs were cloned in pSpCas9(BB)-2A-Puro(pX459) (Addgene #62988) and the activity of sgRNAs at the endogenous target site was initially validated in HEK293T cells using T7 endonuclease I assay52. sgRNA sequences, targets and genotyping primers used in this study are given in the Supplementary Tables 1, 2.

Construction of piggyBac-based donor plasmid

piggyBac DONOR constructs, pDONOR-tagBFP-PSM-EGFP (Addgene #100603) and pDONOR-tagBFP-PSM-dTOMATO (Addgene #100604) were used to introduce sickle cell mutation (SCM) in both alleles. Homology arms were amplified from sickle cell patient-derived genomic DNA and assembled using Gibson isothermal assembly kit (NEB, USA) as per the manufacturer’s instructions on the above mentioned pDONOR constructs, resulting in the final donor vectors, pDONOR-eGFP-SCM and pDONOR-dTomato-SCM. Similarly, to introduce β-thalassemia IVS1-5 mutation (BTM), the homology arms were amplified from a β-thalassemia patient genomic DNA and assembled using the Gibson isothermal method in the piggyBac DONOR constructs. The resultant plasmids were named pDONOR-eGFP-BTM and pDONOR-dTomato-BTM. For ease of screening, a silent mutation was introduced in the right homology arm which generated a new HhaI restriction site. The complete sequences of homology arms and primers for PCR amplification used are provided in Supplementary Tables 3, 4 respectively.

BEL-A culture

BEL-A cells, an immortalized erythroid progenitor cell line (a gift from Profs. Jan Frayne, David Anstee and Dr. Kangtana Trakarsanga, University of Bristol, UK) were cultured11 in serum-free expansion medium (StemSpan SFEM II®, StemCell Technologies) supplemented with 1x Antibiotic Antimycotic solution (Sigma Aldrich), 50 ng/mL human recombinant Stem Cell Factor (Immunotools), 3 IU/ mL Erythropoietin (Peprotech), 1 μg mL Doxycycline (Sigma Aldrich), and 10−6 M Dexamethasone (Sigma Aldrich). The cells were cultured at 37 °C in a 5% CO2 incubator.

Generation of BEL-A SCM and BEL-A BTM erythroid progenitor lines

The disease-specific isogenic lines, BEL-A SCM and BEL-A BTM, were generated from a well-characterized erythroid progenitor cell line BEL-A11 using piggyBac transposase system integrated with CRISPR-mediated genome- editing. 0.5 × 106 BEL-A cells were electroporated with a total of 500 ng plasmid in a ratio of 1:2:2 (pX459-HBBsgRNA: pDONOR-eGFP-SCM or pDONOR-eGFP-BTM: pDONOR-dTomato-SCM or pDONOR-dTomato-BTM) at 1100 V, 30 ms, 3 pulses using the Neon Transfection system (Thermo Fisher Scientific) as per the manufacturer’s protocol. Post nucleofection, when the BFP signal could no longer be detected by flow cytometry, puromycin selection (1 µg/ml) was initiated for the next 72 h. Subsequently, to obtain an enriched subset of potentially biallelic gene-edited cells, eGFP+dTomato+ BFP- cells were sorted. The cell sorting was done in a hierarchical manner, firstly, FSC-A/SSC-A gating was applied to exclude cell debris and acquire target population of cells followed by gating of cells in FSC-W/FSC-H and SSC-W/SS-H to exclude doublets. Subsequently, BFP- cells were gated and amongst them eGFP+ and dTomato+ cells were selected and sorted in BD FACSAriaIII. eGFP+dTomato+BFP- cells were allowed to amplify and were single cell sorted and expanded. On day 15, single cell sorted clones were screened by Flow cytometry, PCR and Sanger sequencing.

Removal of selection cassette using piggyBac transposon

Biallelically targeted clones were used for transposon removal. Transposase treatment was given to BEL-A SCMeGFP+dTomato+ and BEL-A BTMeGFP+dTomato+ mutant lines to remove the selection cassette (positive selection module; PSM) from the integration site. 0.5 × 106 BEL-A mutant cells were mixed with 500 ng of piggyBac transpose expression plasmid (System Biosciences) and electroporated using a Neon electroporation device as described above. The cells were then allowed to amplify. Two weeks post nucleofection dual negative, i.e., SCMeGFP-dTomato-and BTMeGFP-dTomato- cells were sorted separately as one cell/well in 96 well plates.

Genome-editing mediated generation of HPFH beneficial genotypes

CRISPR/Cas9 ribonucleoprotein (RNP) complex was formed by combining 1.5 µg of Cas9 protein (CAS9GFPPRO; Sigma) with 0.5 µg of chemically modified guide RNA (2′-O-methyl analogs and 3′ phosporothioate modifications were included at the three terminal nucleotides of the 5′ and 3′ ends; Sigma) for 10 min at room temperature. For HBG1/2 gene promoter editing, we used 1.5 µg of Cas9 with 0.5 µg of chemically modified sgRNA. For dual sgRNA-mediated HPFH3 genome editing, we used 1.5 µg of Cas9 with 0.25 µg of chemically modified sgRNA for target site-I sgRNA and 0.25 µg of chemically modified sgRNA for target site-II sgRNA. 0.5 × 106 BEL-A WT, BEL-A SCM and BEL-A BTM cells were electroporated using a Neon electroporation device as described above. The electroporated cells were immediately neutralized with a pre-warmed culture medium with cytokines and allowed to recover for 48 h before proceeding to erythroid differentiation.

ddPCR-based quantification of genome editing

The deletion efficiency of the HPFH3 edited BEL-A cells using CRISPR/Cas9 was determined through a ddPCR-based analysis. PCR amplification was done using two sets of primers: (1) the deletion-specific 5BP-F with 3BP-R primers, and (2) the unedited/wildtype-specific 5BP-F with 5BP-R and 3BP-F with 3BP-R primers. The primer information can be found in Supplementary Table 5. The amplified products were further subjected to amplification with EvaGreen® Supermix(BioRad) and the above set of primers using the QX200 BioRad System. The deletion efficiency was calculated using the formula provided.

$${Deletion},{Efficiency}=((D)/(D+W))times 100$$

where,

D is Read count of deletion-specific amplicon

W is Read count of unedited or wildtype specific amplicon

Amplicon sequencing

The editing events and efficiency of the HBG1/2 promoter edited samples were analysed using amplicon based next generation sequencing method53. The PCR amplicons were subjected to fragmentation followed by tagging to adapter sequences (tagmentation). Another round of PCR was performed on the adapter tagged fragments, the adapter index tagged libraries were pooled and cleaned using the purification beads. High Sensitivity dsDNA quantification kit (Invitrogen) was used to quantify the final library. Post normalization and quality assessment the synthesized library was subjected to the paired-end sequencing on Illumina sequencing platform (Illumina Inc.)

For analysis the Fastq files were quality trimmed using Trimmomatic and aligned using bwa mem, further the duplicate reads were removed using picard MarkDuplicates. The resultant BAM file is then analyzed using the R package CrispRVariants for identifying the editing events and efficiency for each sample (Picard, https://broadinstitute.github.io/picard)54,55,56.

Erythroid differentiation of BEL-A cells

The BEL-A WT, disease BEL-A lines and edited BEL-A cells were differentiated into erythroid cells by culturing with differentiation medium of IMDM (Sigma) supplemented with 3% human AB serum (Sigma Aldrich), 2% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific), 1X Antibiotic Antimycotic solution (Sigma Aldrich), 200 µg/mL Holo-transferrin (Sigma Aldrich), 10 µg/mL recombinant human insulin (Sigma Aldrich), 3 IU/mL heparin (STEMCELLTechnologies), 3 IU/mL erythropoietin (Peprotech), 1 µg/mL doxycycline (Sigma Aldrich), 10 ng/mL stem cell factor (Immunotools), 1 ng/mL Interleukin-3 (IL-3) (Immunotools). The cells were seeded at 2 × 105 cells/mL density at day 0 followed by 3.5 × 105 cells/mL on day 2. The cells were further seeded at 5 × 105 cells/mL on day 4 and doxycycline was removed from the medium. From day 6 onwards, 1 × 106 cells/mL density was maintained and the concentration of holo-transferrin was increased to 500 µg/mL. On day 6, SCF and IL-3 were also removed from the medium and the cell density was maintained at 1 × 106 henceforth until Day 1211. At the end of erythroid differentiation, erythroblasts were isolated by centrifuging at 150 g for 5 min.

Erythroid differentiation of CD34+ HSPCs

CD34+ HSPCs were differentiated into mature erythroid cells using the three-phase erythroid differentiation protocol57. During phase 1 (day 0 to day 8), 5 × 104 cells were cultured in an erythroid differentiation medium (EDM) consisting of IMDM (Sigma) supplemented with 100 ng/ml recombinant human SCF, 330 µg/ml human holo-transferrin, 5 ng/ml human IL-3, 3 IU/ml recombinant human erythropoietin (EPO), 2 IU/ml heparin, 5% human AB serum, 20 µg/ml Insulin, 1% GlutaMAX supplement and 1X Antibiotic Antimycotic. During phase 2 (day 9 to 14), cells were seeded in a density of 2 × 105 and cultured in EDM medium without IL-3. During phase 3 (Day 14–21), SCF was also removed from EDM, and cells were cultured at a density of 5 × 105 cells/ml thereafter.

Drug treatment of BEL-A SCM and BEL-A BTM lines

The cells were pre-conditioned by supplementing 50 μM hydroxyurea (H8627; Sigma) and 5 μM Pomalidomide (P0018; Sigma) in BEL-A expansion medium for 4 days. The cells were differentiated as described above, and the drugs were supplemented at the same concentration until day 6. Subsequently, the drugs were removed and differentiation was continued until day 1217.

Flow cytometry analysis for erythroblast characterization

1–3 × 105 cells were harvested on day 12 for BEL-A and Day 21 for HSPCs of erythroid differentiation and stained for differentiation markers anti-CD71-PE (1:100, 60106PE, Clone OKT9, Stem Cell Technologies) and Glycophorin-A-FITC (1:50, 60152FI, clone 2B7, Stem Cell technologies). The cells were incubated with antibodies for 20 min followed by washing with PBS and flow cytometry analysis

For evaluating the efficiency of enucleation, cell-permeable double-stranded DNA dye Hoechst 33342 (2 µg/ml, R37165, Thermo Fisher Scientific) was used according to the manufacturer’s instructions. Briefly, the cells were stained with 2 µg/ml Hoechst 33342 and incubated for 20 min at room temperature. This was followed by washing the cells with PBS and analysis by flow cytometry.

For evaluating the percentage of hemoglobin variants expressed in the total population, 1-3 × 105 erythroid differentiated cells were fixed with 4% formaldehyde (Sigma Aldrich) for 10 min, permeabilized with 0.1% Triton X-100 (Sigma Aldrich) for 5 min. Cells were then washed with PBS supplemented with 2% FBS and stained with anti-HbF-1 (1:50, MHFH05, Thermo Fisher Scientific), anti-hemoglobin β antibody (1:50, sc-21757 FITC, clone 37-8, Santa Cruz Biotechnology) and Anti-hemoglobin α antibody (1:50, sc-514378 PE, clone D-4, Santa Cruz biotechnology). Cells were then washed with PBS and analyzed by flow cytometry.

To determine the reactive oxygen species (ROS) levels, 5 × 105 cells were stained with CM-H2DCFDA (1 µm, C6827, Thermo Fisher Scientific) for 30 min as per the manufacturer’s instructions. Subsequently, the stained cells were washed with PBS and analyzed using flow cytometry.

All the acquisitions were done in BD Accuri C6 analyzer and the data were analyzed in FlowJo software (Version 10). The details of the antibodies and dyes used in the study are compiled in Supplementary Table 6.

Quantitative Real-Time PCR (qRT PCR)

2 × 105 cells were harvested for total RNA isolation on day 7 of BEL-A and day 15 of CD34+ HSPCs erythroid differentiation. RNA was isolated using TRIzolTM (Thermo Fisher Scientific). Total RNA was quantified by spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific). For reverse transcription, 1 µg of total RNA was used for the cDNA synthesis using high-capacity cDNA synthesis kit (Thermo Fisher Scientific) as per the manufacturer’s protocol. The resultant cDNA was used as a template for qPCR with KAPA SYBR Fast (Kapa Biosystems) qRT PCR was done for following genes: HBB, HBA, HBD, HBG, GAPDH, AHSP, BAND3, BCL2L, XPO1, SPTB1, EPOR. In addition, cDNA was used as a template for semiquantitative PCR for erythroid lineage specific Transcription Factors including EKLF, EKLF-3, PABPC1, LMO2, SOX-6, FOG-1, BCL11A, ZBTB7A, GATA-1. The details of all the primers used in the study are mentioned in the Supplementary Table 7.

Giemsa staining

On day 12 of differentiation, 1 × 105 cells were harvested, washed with PBS, resuspended in 30 µL PBS, and smeared onto a clean glass slide. The slide was air-dried, fixed in methanol for 2 min, and then stained with Giemsa (20% in milliQ water) for 5 min, followed by rinsing with milliQ water. The slide was then air-dried, and images were captured using a Brightfield microscope (EVOS M5000, Thermo Fisher Scientific) at 40X magnification.

Reverse phase-high-performance liquid chromatography (RP-HPLC) analysis of Globin chains from human erythroid cells

Globin chain analysis was performed on a reverse phase HPLC system58. Briefly, 1 × 106 BEL-A cells were harvested at day 12 of differentiation and centrifuged at 200 × g for 5 min. Supernatant was discarded and pellet was lysed with the addition of 900 µl of ice-cold MilliQ followed by vortexing and incubation on ice for 20 min. This solution was centrifuged at 9500 x g for 10 min at 4 °C. Supernatant was diluted 1:100 with ice-cold MilliQ and loaded in HPLC vial. 20 µl of the sample was loaded in the column for separation where Buffer A (5% Acetonitrile, 0.1% trifluoroacetic in MilliQ deionized water) was used as loading buffer and Buffer B (95% Acetonitrile, 0.1% trifluoroacetic in MilliQ deionized water) was used as elution buffer.

In vitro sickling assay

On day 12 of differentiation, the cells were incubated for 20 min to form loose red pellets. Supernatant was carefully removed. 1 × 104 cells were resuspended in 150 µL HBSS buffer and seeded in a 96-well plate. The plate was then kept in an incubator (Eppendorf, New Brunswick Galaxy 48 R) at 0.2% oxygen at 37 °C and 5% CO2. After 4 h, BEL-A SCM and BEL-A WT cells derived reticulocytes were assessed for abnormal/sickle shaped morphology using the Floid Cell imaging system at 20X magnification.

Parasite culture

Plasmodium falciparum 3D7 line was thawed and cultured in RPMI-HEPES medium supplemented with 2 g/L sodium bicarbonate (Sigma-Aldrich, USA), 5 g/L Albumax (Invitrogen, USA), 50 mg/L Hypoxanthine (Sigma-Aldrich, USA) and 10 µg/mL gentamicin sulfate (Invitrogen, USA) using human O+ erythrocytes. Culture was maintained at 2% hematocrit at 37 °C under mixed gas conditions (5% CO2, 5% O2, 90% N2).

Invasion assay

Invasion assay was performed with reticulocytes derived from BEL-A SCM and BEL-WT as well as RBCs derived from SCD patients and healthy individuals. For this, P. falciparum 3D7 strain was purified using percoll gradient centrifugation (GE Healthcare) to enrich the 44–46 h schizont stage parasites with a purity of 95%. Purified schizonts were then incubated with the respective erythrocytes for the invasion assay in a culture volume of 100 µL in 96-well plate with the initial parasitemia and hematocrit of 1% and 2% respectively. After 10 h of incubation, slides were prepared and stained with Giemsa (Sigma-Aldrich, USA). Parasites in the ring stage were examined and counted under a light microscope.

Parasite growth assay

To interrogate the effect of parasite growth in reticulocytes derived from BEL-A SCM and BEL-WT as well as RBCs derived from SCD patients and healthy individuals, parasite growth was monitored for a complete life cycle. For this, the parasite was percoll-purified and cultured in 96 wells to maintain the 1% parasitemia and 2% hematocrit with respective erythrocytes/reticulocytes. The development of the parasites was monitored at two different oxygen conditions. In one condition, the culture plate was incubated at 37 °C in a humidified culture chamber along with 0.2 % oxygen while in the other it was maintained at 5% oxygen. Parasites were monitored after every 10 h to examine the parasite growth for the course of one cycle.

TMT labeling, mass spectrometry and data analysis

Sample preparation for proteomics analysis was performed59 on BEL-A BTM, BEL-A SCM, HSPCs BTM, and HSPCs SCM cells. Lysis was done using a 2% lysis buffer (1X Halt Protease and Phosphatase inhibitor cocktail, 2% SDS and 50 mM TEAB) in cold conditions. The protein content in the samples was measured using the PierceTM BCA Protein Assay kit as per the manufacturer’s protocol. Following the BCA estimation, an SDS-PAGE analysis was conducted for quality control. Subsequently, 400 µg of protein was subjected to reduction and alkylation using 10 mM DTT and 20 mM IAA respectively. The protein was precipitated by adding 6X volume of chilled acetone and kept for overnight incubation at −20 °C. 400 µg of protein samples was digested using Trypsin (Promega) in the ratio 1:20 (trypsin: protein) and incubated for 18 hrs at 37 °C. The efficiency of the digestion was assessed by running SDS-PAGE.

The peptide samples were then desalted using SepPak C18 Cartridges. The C18 cartridges were initially activated by passing through 100% Acetonitrile (ACN) and conditioned with 0.1% FA. Later, peptide mixture samples were loaded and passed through the The peptide elutions were used for further processing.

After peptide estimation, 400 µg of the peptides were taken for TMT labeling as per manufacturer’s instructions (Thermo Fisher Scientific). Peptides of BEL-A BTM, BEL-A SCM, HSPCs BTM, and HSPCs SCM were labeled with 127 N, 128 N, 130 N, and 131, respectively. The labeled peptides were fractionated using the basic Reverse Phase Liquid Chromatography (bRPLC) fractionation method using C18 stage tips. A total of 24 fractions were collected and further concatenated to 6 fractions for further LC-MS/MS analysis.

2 µg peptide samples were subjected to mass spectrometer analysis in data-dependent acquisition (DDA) mode using EASY nLC 1200 nano liquid chromatography coupled to Orbitrap Fusion Tribrid (Thermo Scientific, Bremen, Germany) mass spectrometer. Each sample was resuspended with 0.1% FA and loaded onto a 96-well plate. The samples were then loaded onto the Acclaim PepMap™ 100 trap column (75 µm X 2 cm, nanoViper, C18, 3 µm, 100 Å) at a flow rate of 300 nL/min. The peptides were made to pass through and separated using PepMapTMRSLC C18 (2 µm, 100 Å, 50 µm × 15 cm) analytical column. The column equilibration prior to each run and sample loading was performed by passing mobile phase A (0.1% FA). Peptides bound to the C18 were made to separate based on their hydrophobicity and eluted by passing mobile phase A (0.1% FA in Water) and B (0.1% FA in 80% ACN) in gradient mode for 120 min at 300 nL/min flow rate. The %B (80% acetonitrile in 0.1% formic acid) was increased gradually from 5% at 0 min to 100% at 120 min.The column temperature was set to 45 °C throughout the run.

Peptides eluted from the column were ionized in positive ion mode at EASY-Spray™ Source with Spray Voltage: 2.1 kV and Ion Transfer Tube Temp, 275 °C. Ionized peptides were acquired in data-dependent acquisition (DDA) mode with Cycle Time 5 sec. The full MS scan ranged between 400–1600 m/z precursors were acquired in Orbitrap with a resolution of 120 K. The automatic gain control (AGC) and maximum injection time (Max. IT) were set to 2e5 and 20 ms. The precursors were fragmented in the higher energy collision-induced dissociation (HCD) technique with normalized collision energy (NCE) of 35 ± 3%. Finally, the fragment ions were acquired in Orbitrap at 60 K resolution at 200 m/z.

The raw files were searched against the database Homo sapiens v110 (downloaded from NCBI) and the contaminant database in Proteome Discoverer (PD) (version 2.2, Thermo Scientific) software using SEQUEST HT and Mascot search engine. The protease enzyme Trypsin was selected with missed cleavage 1. Carbamidomethylation at Cysteine, TMT-6plex at peptide N-terminus and Lysine were set as fixed modifications. Oxidation at Methionine and Acetylation (N-terminus) were set as the dynamic modifications. Precursor and fragment mass tolerance were set as 10 ppm and 0.02 Da, respectively. The PSMs, peptides and proteins identified with a false discovery rate (FDR) of <5% (q-value < 0.05) were considered true positives. The PD result was quantile normalized and the proteins with fold-change ratios of ≥1.5 and p-value ≤ 0.05 were considered to be significantly upregulated, while those with fold-change ratios of ≤0.66 and p-value ≤ 0.05 were considered to be significantly downregulated.

Statistical analysis

Statistical analysis of data was performed with GraphPad Prism Software (Version 9.5.0 GraphPad Software Inc, USA) using a two tailed student’s t-test. Experiments were performed in triplicates and standard errors of the mean were represented as error bars in all figures. The P-values at p < 0.05 were considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.