Long noncoding RNA HIKER regulates erythropoiesis in Monge’s disease via CSNK2B

Patient samples. All subjects used in this study (CMS and non-CMS) were adult males, lifelong residents of Cerro de Pasco, Peru, and living at an elevation of approximately 4,338 m. CMS patients fulfilled the diagnostic criteria for CMS, or Monge’s disease, based on hematocrit, O2 saturation, and CMS score, as described in detail in our previous studies (6, 52). Sea-level individuals used in this study are individuals who have permanently resided at sea level and are within the age group of CMS and non-CMS subjects.

Native CD34+-derived erythroid cells. Blood samples for PBMC isolation were obtained in sodium heparin–coated tubes. PBMCs were isolated using Histopaque 1077 (Sigma-Aldrich, 10771) by gradient centrifugation. The Dynabeads CD34+ Isolation Kit (Invitrogen, 11301D) was used to purify the CD34+ fraction. CD34+ cells were expanded for a week (days 0–7) in StemSpan medium (STEMCELL Technologies, 09600)containing hydrocortisone (MilliporeSigma, H6909), 50 ng/mL SCF (Peprotech, 300-07), 50 ng/mL FLT3L (Peprotech, 300-19), 10 ng/mL IL-3 (Peprotech, 200-03), 1 ng/mL BMP4 (Peprotech, 120-05), 40 ng/mL IL-11 (Peprotech, 200-11), and 2 U/mL EPO (Amgen, 55513014810). After expansion, cells were further differentiated using the protocol from Giarratana et al. (53). Briefly, cells were then cultured in erythroid differentiation medium (EDM), which includes IMDM supplemented with stabilized glutamine (MilliporeSigma, FG0465), 330 μg/mL holo-human transferrin (MilliporeSigma, T0665), 10 μg/mL recombinant human insulin (MilliporeSigma, I9278), 2 IU/mL heparin, and 5% plasma (Innovative Research, IPLAWBCPD).

iPSC-derived erythroid cells. The iPSC lines from CMS, non-CMS, and sea-level subjects have been generated and well characterized by us (5, 36). The iPSCs were thoroughly assessed using various methods, including DNA fingerprinting, high-resolution karyotyping, and alkaline phosphatase staining, as well as the expression of multilineage differentiation markers, as described in our previous publications (6, 52). We generated the erythroid cultures from iPSCs, following our previously established in vitro platform, based on the protocol of Douay’s group (54). We have previously studied in detail the characteristics of these generated erythroid cells of CMS and non-CMS subjects, including CD markers, maturation, and hemoglobin (6, 54). Briefly, we started the erythroid cultures with approximately 107 to 108 cells of human iPSC cell lines in all subjects. Human iPSCs were differentiated from erythroid cells by formation of embryoid bodies (EBs) for 27 days in a liquid culture medium with the base medium IMDM (MilliporeSigma, FG0465) along with 450 μg/mL holo human transferrin (MilliporeSigma, T0665), 10 μg/mL recombinant human insulin (MilliporeSigma, I9278), 2 IU/mL heparin (NDC 63739-920-25 purchased from McKesson), and 5% human plasma (Innovative Research, IPLAWBCPD) in the presence of 100 ng/mL SCF (Peprotech, 300-07), 100 ng/mL TPO (Peprotech, 300-18), 100 ng/mL FLT3 ligand (Peprotech, 300-19), 10 ng/mL rhu bone morphogenetic protein 4 (BMP4) (Peprotech, 120-05), 5 ng/mL rhu VEGF (Peprotech, 100-20), 5 ng/mL IL-3 (Peprotech, 200-03), 5 ng/mL IL-6 (PeproTech, 200-06), and 3 U/mL Epo (Amgen, 55513014810, purchased from McKesson). This was followed by terminal differentiation as single cells with base medium IMDM (Millipore Sigma, FG0465) along with 5% human plasma (Innovative Research, IPLAWBCPD), 2 IU/mL heparin (McKesson, NDC 63739-920-25), 100 ng/mL SCF (Peprotech, 300-07), 5 ng/mL IL-3 (Peprotech, 200-03), and 3 IU/mL EPO (Amgen, 55513014810).

RNA-Seq and data analysis. Native CD34+ cells were isolated from PBMCs as described above to determine differentially expressed lncRNAs. To do so, RNA was isolated from the erythroid cells after 3 days of exposure to hypoxia or normoxia in CMS (n = 4) and non-CMS (n = 2). RNA was isolated using the Zymo RNA Kit (Zymo, R1050) per the manufacturer’s instructions. The quality of RNA was assessed using TapeStation (Agilent). Ribosome depletion–prepared CMS or non-CMS samples were balanced pooled, and the sequencing libraries were generated by using the TruSeq Stranded Total RNA with RiboZero Gold Library Preparation Kit (Illumina, RS-122-2301). The ribosome-depleted prepared libraries were sequenced using the HiSeq 2500 System in Rapid Run mode (Illumina). A total number of approximately 50 million reads per library were obtained. The resulting reads were mapped using the RUM alignment package with default setting to the human reference hg38. The aligned reads were then processed with htseq-count to obtain the number of reads mapped to genes (Illumina’s iGenome GTF annotation for hg38). Quality control (QC) processes were performed prior to and after alignment to ensure high quality of final results. This included GC content, the presence of adaptors, FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for sequence quality, overrepresented k-mers, and duplicated reads, and Picard (http://broadinstitute.github.io/picard/)/RseQC for mapping quality. Differentially expressed transcripts were determined by EBSeq (55). LNCipedia (https://lncipedia.org/) and GENCODE (https://www.gencodegenes.org/) were used for lncRNA annotation (19, 5665).

To determine DEGs following HIKER/LINC02228-KD or LINC00431-KD, total RNA was isolated from the CMS iPSC-derived CD34+ with or without a KD of HIKER/LINC02228 or LINC00431 using the Zymo RNA Kit (Zymo, R1050), and the RNA-Seq libraries were generated using the Illumina TruSeq Stranded Total RNA Kit (Illumina, catalog RS-122-2301) per the manufacturer’s instructions. A total of more than 40 million reads per library were obtained following sequencing with the HiSeq 2500 System. After QC, the resulting reads were mapped using the RUM alignment package with default setting to the human reference hg38. Differentially expressed transcripts were determined by DESeq2 (66).

Cellular fractionation and qPCR analysis of differentially expressed lncRNAs. Briefly, total nuclear and cytoplasmic extracts were isolated from erythroid cultures (iPSC-derived CD34+ cells isolated from EBs as described in detail above) using Active Motif (catalog 40010) according to the manufacturer’s instructions. qPCR for HIKER/LINC02228, LINC01133, APOBEC3B-AS1, UBE2Q-AS1, and LINC00431 were used to assess the purity of the fractions. Primers are listed in Supplemental Table 3.

KD of nuclear lncRNA HIKER/LINC02228 and LINC00431 expression using QIAGEN LNA gapmers ASO. Locked nucleic acids (LNAs) targeting HIKER/LINC02228 and LINC00431 were designed and synthesized by Exiqon. Detailed sequences are listed in Supplemental Table 3. The most efficient ASO for each LNA was initially tested in the pilot experiment with and without transfection reagent (Lipofectamine 3000, Life Technologies, L3000-008) in a dose-response experiment at a concentration of 10 nM, 25 nM, 50 nM, and 100 nM. The uptake and the effect of ASO were monitored by qPCR at various stages (iPSC stage and CD34+ cells isolated from EBs). For both lncRNAs, the optimal delivery for all the stages was at the 50 nM concentration without the transfection reagent.

Isolation of CD34+ cells from iPSC-derived EBs. CD34+ cells were isolated from iPSC-derived EBs as follows. After 7 days of differentiation, EBs were harvested by spinning at 400g for 10 minutes. After centrifugation, EBs were dissociated into single cells using Accutase treatment for 10 minutes and then filtered through a 60 μm cell strainer (Falcon). CD34+ cells were isolated from this cell suspension using EasySep Human CD34 Positive Selection Kit II (STEMCELL Technologies, 17856) per the manufacturer’s instructions. These iPSC-derived CD34+ cells were used in subsequent qPCR and colony-forming assays.

BFU-E and CFU-E assays. CD34+ cells used in this assay were derived from iPSC-generated EBs as described above. CD34+ cells were plated at a density of 105 cells per 35 mm dish combined with MethoCult H4034 Optimum Media (STEMCELL Technologies, 04044) and 2% FBS. Dishes were incubated at 37°C in an incubator with 5% CO2 and 5% O2 for 14 days, at which time colonies were scored for BFU-E and CFU–granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM).

KD and OE constructs for CSNK2B and lentiviral transduction. KD lentiviral particles were purchased from Santa Cruz Biotechnology Inc., and OE construct and lentiviral particles were generated by Vector Builder. The iPSCs from CMS and non-CMS cells were transduced with polybrene (8 μg/mL, MilliporeSigma, TR-1003-G) at MOI within the range of 1 to 5 (with the titer of lentivirus ranging from 107 to 109). The optimal concentration was determined for the transduction and antibiotic selection by performing dose-specific kill curves. Transduced cells were selected at 0.5 μg/mL puromycin (Sigma-Aldrich, 58-58-2) or 0.5 μg/mL blasticidin (EMD Millipore, 20-335). For double KD, puromycin and blasticidin combinations were used for selection. The expression of CSNK2B in each construct was verified by qPCR at the iPSC stage as well as the iPSC-derived CD34+ stage.

In vitro casein kinase inhibitor experiments. TBB (catalog ab120988) and CX4945 (catalog S2248) were purchased from Abcam and Selleckcam, respectively. Dose-response experiments were performed with the inhibitors using the following concentrations in the colony forming assays using iPSC-derived CD34+ cells as described above: TBB (25 μM, 50 μM, and 100 μM) and CX4945 (2.5 μM, 5 μM, and 10 μM).

Western blot analysis for quantification of protein levels. Proteins were isolated using standard protein isolation protocols with RIPA buffer (Cell Signaling Technology, 9806) and protease inhibitor cocktail (Roche, 11697498001). For protein isolation, EBs at week 1 were used in this study. Through FACS analysis, we determined that at this stage, the population of erythroid cells was at the CD34+ stage. Antibodies against CSNK2B (Abcam, catalog ab76025), DXO (Abcam, catalog ab152135), PPP1R11 (Abcam, catalog ab171960), ZNRD1 (Santa Cruz Biotechnology Inc., catalog sc-393406), and TAP2 (Santa Cruz Biotechnology Inc., catalog sc-515576) were purchased. At the same protein concentration, GAPDH (Cell Signaling Technology, catalog 2118S) was used as the control for normalizing during quantification of the blots. In brief, 20 μg of lysate supernatant was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The blots were developed using enhanced chemiluminescent reagents (Bio-Rad Laboratories) and the ChemiDoc XRS+ Molecular Imager (Bio-Rad Laboratories).

Zebrafish husbandry and maintenance. Zebrafish (Danio rerio) were raised in a circulating aquarium system on a 14-hour light/10-hour dark cycle at 28.5°C, following standard husbandry procedures (67).

Morpholino and mRNA microinjection. The morpholino antisense oligo (MO) 5′-CGACACTTCCTCTGAGCTACTCATG-3′ was synthesized to block the translation initiation of csnk2b, and the 5-mismatch oligo 5′-CGAGAGTTCGTCTGACCTAGTCATG-3′ was synthesized as a specificity control (Gene Tools). For synthesizing csnk2b rescue mRNA that is resistant to the translation blocking MO, the full-length csnk2b coding sequence with 4 base pairs of silent mutations in the MO recognition region was cloned into the pCS2-vector (Azenta Life Sciences), in which the first 24 base pairs of the csnk2b coding sequence became 5′-ATGAGTAGCTCAGAAGAGGTCTCC-3′. The csnk2b capped mRNA was synthesized using the mMESSAGE mMACHINE Kit (Ambion, AM1340). Microinjection was performed on WT AB embryos at the 1- to 2-cell stages. Unless otherwise indicated, each embryo was injected with 5 ng of csnk2b MO and 50 pg of csnk2b mRNA for KD and rescue, respectively.

Hemoglobin staining. Embryos at 2 dpf were dechorionated and anesthetized with 0.016% tricaine (Fluka, A5040), followed by a 15-minute incubation in 0.6 mg/mL o-dianisidine solution (Sigma-Aldrich, D9143). This solution was prepared in 0.65% H2O2(EMD, HX0647-3), 40% ethanol (KOPTEC, 89125), and 10 mM sodium acetate (Fisher Chemical, S210-500) at room temperature. Stained embryos were washed twice with 1× PBS (Gibco, Thermo Fisher Scientific, 14200166) and then fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich, P6148). Hemoglobin signal was observed under a light microscope and quantified according to the area and intensity in the heart and common cardinal vein; embryos were categorized into normal, medium, and low hemoglobin levels.

Data availability. RNA-Seq data were deposited in the NCBI’s Sequence Read Archive (SRA BioProject PRJNA826881).

Statistics. For qPCR analysis under hypoxia and normoxia conditions, 2-tailed, unpaired t tests were performed. For multiple comparisons, such as for the inhibitor experiments or BFU assays, we performed 1-way ANOVA followed by Tukey’s tests. For in vivo study, χ2 tests were performed to examine the statistical differences of the phenotype distribution between groups. P < 0.05 was considered statistically significant.

Study approval. For human studies, each subject provided informed, written consent under IRB protocols approved by UCSD and the Universidad Peruana Cayetano Heredia, Lima, Peru. For zebrafish studies, animal protocols were approved by the UCSD Animal Research Committee to ensure compliance with all tenets of the Animal Welfare Act and Public Health Service (PHS) policy.

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