Novel differential calcium regulation of hematopoietic stem and progenitor cells under physiological low oxygen conditions

Low oxygen bone marrow (BM) niches (~1%–4% low O2) provide critical signals for hematopoietic stem/progenitor cells (HSC/HSPCs). Our presented data are the first to investigate live, sorted HSC/HSPCs in their native low O2 conditions. Transcriptional and proteomic analysis uncovered differential Ca2+ regulation that correlated with overlapping phenotypic populations consisting of robust increases of cytosolic and mitochondrial Ca2+, ABC transporter (ABCG2) expression and sodium/hydrogen exchanger (NHE1) expression in live, HSC/HSPCs remaining in constant low O2. We identified a novel Ca2+ high population in HSPCs predominantly detected in low O2 that displayed enhanced frequency of phenotypic LSK/LSKCD150 in low O2 replating assays compared to Ca2+ low populations. Inhibition of the Ca2+ regulator NHE1 (Cariporide) resulted in attenuation of both the low O2 induced Ca2+ high population and subsequent enhanced maintenance of phenotypic LSK and LSKCD150 during low O2 replating. These data reveal multiple levels of differential Ca2+ regulation in low O2 resulting in phenotypic, signaling, and functional consequences in HSC/HSPCs.

Regulation of Ca 2+ occurs at the cytosolic, mitochondrial, and nuclear levels, is dependent upon voltage gated channels/exchangers (sodium-hydrogen exchanger 1/NHE-1), plasma membrane efflux pumps/ABC transporters (ABCG2) and is sensitive to changes in pH (Bunting 2002;Cheng et al., 2019;Krishnamurthy et al., 2004;Li et al., 2013;Nakamura et al., 2008;Slepkov et al., 2007;Stewart et al., 2015;Tang et al., 2010). Ca 2+ regulation has most commonly been studied in the context of plasma membrane Ca 2+ efflux pumps (PMCA) and sodium/Ca 2+ exchangers (NCX). In HSCs, PMCAs play a role in the lower intracellular Ca 2+ levels noted in HSCs compared to more committed progenitors due to increased PMCA activity in HSCs (Luchsinger et al., 2019). ABC transporters are most importantly known for their role in multidrug resistance with the ability to pump substances (chemotherapeutics, toxic substances, chemical modulators, etc.) across the cell membrane as well as ABCG2 acting as a highly expressed marker of stem cell (side) populations (Goodell et al., 1996;Scharenberg et al., 2002;Zhou et al., 2001). Other links between ABC transporters and Ca 2+ regulation include ABCG1 enhancement of the hypotonicity-induced Ca 2+ response (induced by intracellular Ca 2+ release from the ER) seen in HEK293 cells (Dunn et al., 2020). ABCG3 has been shown to be regulated in a Ca 2+ -dependent manner in MDA-MB-468 breast cancer cells (Stewart et al., 2015) and previous studies identified a correlation between plasma membrane efflux pump ABCG2 (CD338) and Ca 2+ showing the presence of a Ca 2+ antagonist in HeLa cells led to decreased transport activity of ABCG2 (Takara et al., 2012).
Ca 2+ regulation and impacts can occur directly or indirectly.
Protons, like Ca 2+ , serve as a second messenger regulating proliferation, differentiation, and cell death (Schreiber 2005;Slepkov et al., 2007). pH regulation is controlled by exchangers including NHE1. NHE1, is ubiquitously expressed and in HSCs has been studied in the context of hematological malignancy and alterations to intracellular pH (Hyun et al., 2019;Man et al., 2022;Rich et al., 2000) with extracellular pH alterations being linked to alterations in Ca 2+ signaling (Nakamura et al., 2008). At homeostasis, NHE-1 limits early influx of sodium into the cell preventing intracellular Ca 2+ overload via coupling with NCX. Upon activation, NHE1 sodium flux increases and induces intracellular Ca 2+ overload (Nakamura et al., 2008).
Unfortunately, although regulation of HSC/HSPCs in low O 2, as well as the regulations and roles of Ca 2+ in HSC/HSPC function, have been independently investigated in the literature, the studies identifying these insights have all been performed in air, with cells exposed to air/nonphysiologic conditions, stress conditions, or using whole BM/fixed cells. Thus, leaving a gap in our understanding of HSC cellular physiology and potentially leaving endogenous HSC/ HSPC low O 2 regulatory pathways, including Ca 2+ and potential transporters altered, unidentified, or unclear. Our current novel investigations are the first to compare live, sorted HSC/HSPCs isolated/retained in low O 2 to their identical counterparts exposed to air, as previously the technology to do so did not exist. Our investigations of Ca 2+ regulation in HSC/HSPCs in continuous low O 2 , have identified unique differential Ca 2+ regulation at transcriptional and protein levels leading to phenotypic/functional attributes in low O 2 HSC/HSPCs that have not been observed in air. This substantiates that differential regulation of Ca 2+ is occurring endogenously at multiple levels in low O 2, including but not limited to, increased expression of sodium/hydrogen exchangers (NHE1) and plasma membrane efflux pumps (ABCG2). We identified increased, and overlapping, ABCG2 expression and cytosolic/mitochondrial Ca 2+ influx in HSC/HSPCs including novel Ca 2+ high (hi) subpopulations (in low O 2 ) that displayed enhanced stem cell maintenance, decreased rate of differentiation, and preserved stem cell phenotype in functional replating assays more efficiently than their Ca 2+ low (lo) counterparts. Together, these findings reveal endogenous unique populations, signaling, and roles for differential Ca 2+ regulation, in low O 2 in HSCs and HSPCs. Cells were then split into ambient air or retained in low O 2 (60 min).

| Animals
All data was analyzed using FlowJo9.6 (TreeStar Inc. The reads were mapped to the mouse genome mm10 using STAR (v2.5) (Dobin et al., 2013). RNA-seq aligner with the following parameter: "--outSAMmapqUnique 60." Uniquely mapped sequencing reads were assigned to gene using featureCounts (v1.6.2) (Liao et al., 2014) with the following parameters: "-s 0 -p -Q 10 -O." The data was filtered using read count >10 in at least two of the samples, normalized using TMM (trimmed mean of M values) method and subjected to differential expression analysis using edgeR (v3.20.8) (McCarthy et al., 2012;Robinson et al., 2010). Gene ontology and KEGG pathway functional analysis was performed on differential expression gene using DAVID (Database for Annotation, Visualization and Integrated Discovery) (Dennis et al., 2003;Huang et al., 2009). Ttest statistical analysis was utilized for analysis of differential expression data. Differential expression genes in HSPCs (LSK) samples were determined using false discovery rate adjusted p value cut-off of 0.05. Differential expression genes in HSC (LSKCD150+) samples were determined using p value cut-off of 0.01 and FDR of 0.05.

| Proteomics analysis
Due to the low cell numbers required for this technique (~150k) and based on insight gained from initial samples, LSK cells were sorted in low O 2 , and either exposed to ambient air or remained in low O 2 (60 min). Samples were lysed in their ambient air or low O 2 using 5% After each addition, the sample was centrifuged at 4000g, 4°C for 3 min and the flow-through was transferred to a sample vial.
Peptides were then dried in a speed-vac. The dried peptides were resuspended with 20 µL of 50 mM acetic acid and the concentration was determined by absorbance at 205 nm. The timsTOF Pro was operated in PASEF mode (Meier et al., 2015(Meier et al., , 2018. Mass spectra for MS and MS/MS were recorded over the m/z range 100-1700 using a spray voltage of 1500 V. Ion mobility resolution was set to 0.6-1.6 V × s/cm. with data dependent acquisition was performed with 10 PASEF scans per cycle. A polygon filter was applied in the m/z and ion mobility space to exclude low m/z, singly charged ions from PASEF precursor selection. Active exclusion was applied, releasing after 0.4 min, with precursor reconsidered if current intensity/previous intensity was 4.0 or greater.

| Proteomics data analysis
The resulting mgf files were searched using Mascot Daemon by Results were filtered to 1% false discovery rate and a minimum of two peptides per protein identification. Total normalized spectral counts were used to quantify relative protein abundances. T-test was used for statistical analysis of results.

| Statistics
All bioinformatics data were analyzed as stated in above sections.
Statistical analysis between two groups was performed using a twotailed paired Student's t-test. When more than two groups were compared, one-way analysis of variance using Bonferroni post hoc analysis was used. Differences between group means were considered significant when the probability value p < 0.05. Biological replicates are represented by the samples size "n" for each figure and all experiments were performed at least twice with at least an n of four per experiment unless otherwise noted. Statistical data was computed using both Prism 6 (GraphPad) and Excel (Microsoft Office).

| RESULTS
3.1 | Identification of differential transcriptional and translational Ca 2+ regulation in native low O 2 Our novel technology allowed us to be first to initiate studies directly comparing live, isolated HSC/HSPCs in their native low O 2 environment, never exposed to air. All experimental samples were harvested, isolated, and sorted under low O 2 and either remain in low O 2 (never exposed to air) or are split off from the original identical sample and exposed to air (Figure 1a). To expand our understanding of the endogenous low O 2 signaling of HSC/HSPCs, we performed bulk mRNA sequencing on live, sorted LSK (Lin − Sca1 + c-KIT + ) and LSKCD150 + (Lin − Sca1 + c-KIT + CD150 + ) populations either remaining in low O 2 or exposed to air (Figure 1a).
In sorted LSK cells, bulk mRNA analysis (23,611 total genes) identified 1239 differentially expressed genes (DEGs, data not shown).
The   (Figure 3a,b) and significant increases in the percentage of Rhod-2 positive cells (Figure 3c) in HSCs and HSPCs in low O 2 compared to identical populations exposed to air.

| HSPCs and HSCs exhibit enhanced expression of ABC transporter ABCG2 and overlap with Ca 2+ populations in low O 2
Ca 2+ regulation is dependent upon various pathways/mechanisms including expression of plasma membrane efflux pumps (which were also enriched in our transcriptional data). Although Ca 2+ regulation via plasma membrane pumps has been most extensively studied in the context of PMCAs, ABC transporters have also been shown to influence Ca 2+ regulation. ABCG2, highly expressed in HSCs and a known HSC stem cell marker, has been linked to Ca 2+ regulation showing the presence of a Ca 2+ antagonist in HeLa cells led to decreased transport activity of ABCG2 in air (Scharenberg et al., 2002;Takara et al., 2012;Zhou et al., 2001). After noting enhancements in plasma membrane efflux pumps and transport activity in our multiomics data, we examined ABCG2 in HSCs and HSPCs and its potential role in Ca 2+ regulation. We identified increases in the total number and percentage of ABCG2 + cells in HSPCs and HSCs in low O 2 compared to identical samples exposed to air (Figure 4a). O 2 overlap between cytosolic/mitochondrial Ca 2+ and ABCG2 expression was enhanced in comparison to the overlap in air exposed samples. As expected, based on the barely distinguishable Fura Hi cells identified in air compared to in low O 2 , HSCs (12-fold) and HSPCs (3.8-fold) with both high ABCG2 expression and Ca m 2+ also demonstrated an increase in the Fura Hi population in low O 2 compared to air (Figure 4j,k).
The culmination of these data reveal that ABCG2 surface expression is enhanced in low O 2 in HSC/HSPCs with phenotypic crossover and a strong relationship/regulation between cytosolic/ mitochondrial Ca 2+ and ABCG2 surface expression in HSCs and HSPCs in low O 2 , compared to air.
3.5 | Inhibition of NHE1 blunts enhanced Ca 2+ flux and maintenance of HSC/HSPC phenotype in low O 2 Our transcriptional/proteomic analyses highlighted differential regulation of Ca 2+ and transmembrane transporters which was confirmed by phenotypic/functional analyses demonstrating enhancements in intracellular Ca 2+ and ABCG2 expression in HSC/HSPCs in native low O 2 (Figures 1-4). type, function, and frequency compared to air exposed cells.
Inhibition of NHE1 blunts these enhancements and subsequently leads to loss of the enhanced Fura Hi population.

| DISCUSSION
The BM niche provides critical support for HSC/HSPCs. Factors such as O 2 levels/microenvironmental alterations, niche interaction, and modification of metabolic regulation all impact normal hematopoiesis with their disruption potentially leading to senescence/dysfunction (Mantel et al., 2015;Mohyeldin et al., 2010;Spencer et al., 2014;Suda et al., 2011;Takubo & Suda 2012;Zhang et al., 2012). Previous studies exposed whole BM harvested in low O 2 to air for as little as 10 min resulting in diminished numbers of phenotypic HSC/HSPCs (Mantel et al., 2015).   Hoechst 33342 staining, and expression alone, have also been shown to be a marker for HSC/HSPCs (Bunting 2002;Goodell et al., 1996;Norwood et al., 2004;Stewart et al., 2015;Tang et al., 2010;Zhou et al., 2001). We demonstrate enhanced ABCG2 expression in low O 2 in HSCs and HSPCs greater than previously reported in air studies inhibition with Cariporide (CARP, Figure 5i,j). Based on these insights as well as this being the first study to observe LSK and LSKCD150 cells that have not been exposed to ambient air, it is tempting to speculate that not only are the expression and activity of multiple transporters likely differentially regulated in low O 2 with potential redundant functions, but that they may be quickly turned on and off uniquely in low O 2 to optimize desired Ca 2+ homeostasis while playing other important endogenous roles yet to be fully elucidated. These observations of differential