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• W4328049861 abstract "Report21 March 2023Open Access Transparent process pH regulates hematopoietic stem cell potential via polyamines Sachin Kumar Corresponding Author Sachin Kumar [email protected] orcid.org/0000-0002-3835-8935 Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, India Contribution: Conceptualization, Data curation, Formal analysis, Supervision, ​Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Jeffrey D Vassallo Jeffrey D Vassallo orcid.org/0000-0002-4090-3779 Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA Contribution: Conceptualization, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Kalpana J Nattamai Kalpana J Nattamai Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA Contribution: Data curation, Formal analysis, ​Investigation, Visualization, Methodology, Project administration Search for more papers by this author Aishlin Hassan Aishlin Hassan orcid.org/0000-0003-4427-3564 Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA Contribution: Data curation, Formal analysis, ​Investigation, Visualization, Methodology, Project administration, Writing - review & editing Search for more papers by this author Rebekah Karns Rebekah Karns Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center and University of Cincinnati, Cincinnati, OH, USA Contribution: Data curation, Formal analysis Search for more papers by this author Angelika Vollmer Angelika Vollmer Institute of Molecular Medicine, Ulm University, Ulm, Germany Contribution: Formal analysis, ​Investigation, Visualization, Methodology Search for more papers by this author Karin Soller Karin Soller Institute of Molecular Medicine, Ulm University, Ulm, Germany Contribution: ​Investigation Search for more papers by this author Vadim Sakk Vadim Sakk Institute of Molecular Medicine, Ulm University, Ulm, Germany Contribution: ​Investigation Search for more papers by this author Mehmet Sacma Mehmet Sacma orcid.org/0000-0003-3450-674X Institute of Molecular Medicine, Ulm University, Ulm, Germany Contribution: Data curation, Software, Formal analysis, ​Investigation, Visualization, Methodology Search for more papers by this author Travis Nemkov Travis Nemkov University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, USA Contribution: Data curation, Formal analysis, ​Investigation, Methodology Search for more papers by this author Angelo D'Alessandro Angelo D'Alessandro orcid.org/0000-0002-2258-6490 University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, USA Contribution: Resources, Data curation, Formal analysis, Supervision, ​Investigation Search for more papers by this author Hartmut Geiger Corresponding Author Hartmut Geiger [email protected] orcid.org/0000-0002-5794-5430 Institute of Molecular Medicine, Ulm University, Ulm, Germany Aging Research Center, Ulm University, Ulm, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Sachin Kumar Corresponding Author Sachin Kumar [email protected] orcid.org/0000-0002-3835-8935 Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, India Contribution: Conceptualization, Data curation, Formal analysis, Supervision, ​Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Jeffrey D Vassallo Jeffrey D Vassallo orcid.org/0000-0002-4090-3779 Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA Contribution: Conceptualization, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Kalpana J Nattamai Kalpana J Nattamai Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA Contribution: Data curation, Formal analysis, ​Investigation, Visualization, Methodology, Project administration Search for more papers by this author Aishlin Hassan Aishlin Hassan orcid.org/0000-0003-4427-3564 Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA Contribution: Data curation, Formal analysis, ​Investigation, Visualization, Methodology, Project administration, Writing - review & editing Search for more papers by this author Rebekah Karns Rebekah Karns Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center and University of Cincinnati, Cincinnati, OH, USA Contribution: Data curation, Formal analysis Search for more papers by this author Angelika Vollmer Angelika Vollmer Institute of Molecular Medicine, Ulm University, Ulm, Germany Contribution: Formal analysis, ​Investigation, Visualization, Methodology Search for more papers by this author Karin Soller Karin Soller Institute of Molecular Medicine, Ulm University, Ulm, Germany Contribution: ​Investigation Search for more papers by this author Vadim Sakk Vadim Sakk Institute of Molecular Medicine, Ulm University, Ulm, Germany Contribution: ​Investigation Search for more papers by this author Mehmet Sacma Mehmet Sacma orcid.org/0000-0003-3450-674X Institute of Molecular Medicine, Ulm University, Ulm, Germany Contribution: Data curation, Software, Formal analysis, ​Investigation, Visualization, Methodology Search for more papers by this author Travis Nemkov Travis Nemkov University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, USA Contribution: Data curation, Formal analysis, ​Investigation, Methodology Search for more papers by this author Angelo D'Alessandro Angelo D'Alessandro orcid.org/0000-0002-2258-6490 University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, USA Contribution: Resources, Data curation, Formal analysis, Supervision, ​Investigation Search for more papers by this author Hartmut Geiger Corresponding Author Hartmut Geiger [email protected] orcid.org/0000-0002-5794-5430 Institute of Molecular Medicine, Ulm University, Ulm, Germany Aging Research Center, Ulm University, Ulm, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Sachin Kumar *,1,2, Jeffrey D Vassallo1, Kalpana J Nattamai1, Aishlin Hassan1, Rebekah Karns3, Angelika Vollmer4, Karin Soller4, Vadim Sakk4, Mehmet Sacma4, Travis Nemkov5, Angelo D'Alessandro5 and Hartmut Geiger *,4,6 1Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA 2Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, India 3Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center and University of Cincinnati, Cincinnati, OH, USA 4Institute of Molecular Medicine, Ulm University, Ulm, Germany 5University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, USA 6Aging Research Center, Ulm University, Ulm, Germany *Corresponding author. Tel: +91 522 2772550 ext 4873; E-mail: [email protected] *Corresponding author. Tel: +49 731 50 26700; Fax: +49 731 50 26710; E-mail: [email protected] EMBO Reports (2023)24:e55373https://doi.org/10.15252/embr.202255373 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Upon ex vivo culture, hematopoietic stem cells (HSCs) quickly lose potential and differentiate into progenitors. The identification of culture conditions that maintain the potential of HSCs ex vivo is therefore of high clinical interest. Here, we demonstrate that the potential of murine and human HSCs is maintained when cultivated for 2 days ex vivo at a pH of 6.9, in contrast to cultivation at the commonly used pH of 7.4. When cultivated at a pH of 6.9, HSCs remain smaller, less metabolically active, less proliferative and show enhanced reconstitution ability upon transplantation compared to HSC cultivated at pH 7.4. HSCs kept at pH 6.9 show an attenuated polyamine pathway. Pharmacological inhibition of the polyamine pathway in HSCs cultivated at pH 7.4 with DFMO mimics phenotypes and potential of HSCs cultivated at pH 6.9. Ex vivo exposure to a pH of 6.9 is therefore a positive regulator of HSC function by reducing polyamines. These findings might improve HSC short-term cultivation protocols for transplantation and gene therapy interventions. Synopsis Culture conditions that maintain HSC potential ex vivo are of high clinical interest. This study shows that the potential of murine or human HSCs is maintained for 2 days by reducing polyamines when cultivated at a pH of 6.9. The pH in the bone marrow can be lower than the serum pH of 7.4 and can be as low as pH 6.4 in pH pockets. Two-day cultivation of HSCs at a pH of 6.9 results in the maintenance of murine or human HSC functions. A change in pH from 7.4 to 6.9 alters the metabolism of HSCs, including attenuation of the polyamine pathway. Blocking polyamine synthesis in HSCs by DMFO recapitulates maintenance of murine or human HSCs at pH 7.4. Introduction Blood cells are sustained by hematopoietic stem cells (HSCs) in a process termed hematopoiesis (Seita & Weissman, 2010; Mendelson & Frenette, 2014). Genetic manipulation of HSCs ex vivo, usually coupled to a subsequent transplantation, has demonstrated a therapeutic benefit in the treatment of monogenetic diseases like bone marrow failures or to generate the next generation of CAR-T cells (Zhen et al, 2021). Understanding the mechanisms that regulate the function of HSCs ex vivo is therefore of high therapeutic value, as a limitation of a broader therapeutic use of HSCs remains the difficulty to maintain their potential ex vivo (Kumar & Geiger, 2017). Current clinical protocols for ex vivo culture of HSCs usually lead to a loss of stemness and increase in differentiation potential. There is therefore a therapeutic need for novel approaches to at least maintain HSC function upon ex vivo culture. Interestingly, changes in pH can affect the relative output of erythroid, granulocyte, and megakaryocytic cells upon differentiation of CD34+ human hematopoietic progenitor cells ex vivo (McAdams et al, 1997, 1998; Hevehan et al, 2000; Yang et al, 2002). Within the bone marrow (BM) niche, a mild extracellular acidosis can be accompanied by changes in pH due to the accumulation of lactic acid following anaerobic metabolism of HSCs (Simsek et al, 2010; Norddahl et al, 2011; Wang et al, 2014), neutrophils (Khatib-Massalha et al, 2020), or in leukemic conditions (Padda et al, 2021). pH might therefore be a physiological modifier of HSC differentiation, and likely thus also for HSC potential. While the effect of pH on HSC function has not been investigated so far, very recent publications imply a role for changes intracellular pH for epithelial cell plasticity and embryonic stem cell differentiation (Ulmschneider et al, 2016; Liu et al, 2020). Here we demonstrate that an extracellular pH of 6.9 in ex vivo culture, which is distinct from the commonly used pH of 7.4, results in maintenance of HSCs ex vivo and affects HSC size and stress. Metabolite analyses identified the polyamine pathway as a target in pH 6.9 HSCs. Pharmacological inhibition of polyamine synthesis maintained HSC potential ex vivo even at a pH of 7.4. Collectively, these results establish extracellular pH and the polyamine pathway as a regulator of the function of HSCs ex vivo, which may be further leveraged to improve ex vivo cultivation protocols for clinical applications. Results and Discussion Bone marrow pH is lower than in blood HSCs reside within BM in specialized regions termed niches. Previous reports suggest there are, upon disease, tissue-specific levels of pH which are distinct from blood (Reshetnyak et al, 2020). The pH range HSCs are exposed to in vivo is not known. Therefore, initial experiments were conducted to determine the pH within murine BM. Bone cavity showed a pH of 7.2, which is lower compared to the pH in blood (pH 7.4; Figs 1A, and EV1A and B). Additional studies using multiphoton live microscopy and a fluorescent pH-sensitive ratiometric probe, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, Fig EV1C and D; Ray et al, 2012) demonstrated that the distribution of pH within BM is not uniform and heterogeneous (Fig EV1E and F). The analysis of selective landmarks as regions of interest (ROI) at either the bone surface or within BM including cells with likely osteoblastic and osteoclastic nature or known blood vessels (Fig EV1E and G) revealed that pH values within the BM of young mice might range from pH 6.4 to a pH of 7.8. HSC might thus, in their natural environment, be continuously exposed to a level of pH that is lower than pH 7.4, which is usually used in ex vivo culture medium. There is even the possibility that HSCs might reside in distinct pH niches in bone marrow. To the best of our knowledge, though it is currently not feasible to determine the position of HSCs together with the level of pH at that position in BM. Figure 1. A pH of 6.9 in the medium maintains HSC function A. pH within bone marrow in mice measured by using a 0.6 mm combination pH probe that was well calibrated with 4.00, 7.00, and 10.00 standards with > 0.98 R2 reading. n = 5 or more animals from two independent experiments. Data were analyzed using two-tailed Student's t-test, and exact P values are mentioned between groups. B. HSC (100 cells) from C57BL/6 mice (Ly5.2) were cultured under distinct pH (6.9–7.8) settings for 40 h and transplanted alongside 2 × 105 competitor cells from BoyJ mice (Ly5.1). Summary of initial screening of diverse pH effects on contribution of total donor-derived Ly5.2+ cells in PB and BM in competitive primary and secondary transplants. Data are means of n = at least 14 mice per group and five experiments. C–J. HSC (100 cells) were untreated or cultured under pH 7.4 and pH 6.9 for 40 h and transplanted with competitor cells. n = at least 18 mice per group out of at least seven experiments (biological repeats) (C, F) Contribution of donor-derived Ly5.2+ cells to PB cells in primary (C) and secondary (F) recipients between 4 and 20 weeks post transplant. **P < 0.01, ***P < 0.001 with respect to pH 7.4 treatment or between marked groups using two-tailed Student's t-test. (D, G) Relative contribution of T cells, B cells, and myeloid cells among PB donor-derived Ly5.2+ cells in primary (D) and secondary (G) recipients at 16–20 weeks post transplant. (E, H) Contribution of donor-derived Ly5.2+ cells to BM cells in primary (E) and secondary (H) recipients at 16–20 weeks post transplant. **P < 0.01 using two-tailed Student's t-test. (I, J) Representative FACS dot plots and quantitative and statistical analysis of LT-HSC, ST-HSC, and LMPP distribution among donor-derived LSKs in primary (I) and secondary (J) recipients. *P < 0.05 using two-tailed unpaired Student's t-test. Data information: All data are means ± SE. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. pH probe for BM pH measurement in vivo, media pH calibration A combination pH probe (0.6 mm in diameter) from Specialty Sensor LLC, USA was utilized. The picture depicts the size of the probe relative to a pencil lead point and the stand for calibration with aluminum cover to avoid any interference. Standard curve calibrated with 4.00, 7.00, and 10.00 standards. Data represent n = 3 technical replicates. Data are means ± SE. Representative 2-photon excitation spectra of the pH-sensitive probe, 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) from 700 to 950 nm at different pHs. Standard curve using the ratio of the pH-sensitive 850 nm and the pH-neutral 750 nm spectra of HPTS against pH at a concentration of 1 mM and 100 μM HPTS. Data are means ± SE. Representative of n = 3 technical replicates. 2-photon analysis of pH-sensitive probe, left side image depicts HPTS at 850 nm (green) in the bone marrow cavity of tibia in live animals. Blood vessels were labeled with dextran TRITC (red), while the bone surface was identified by a standard second harmonic generation approach (blue) (scale bar = 50 μm). Right side image depicts the ratio of HPTS intensity at 850 and 750 nm. Arrows (1–3) represent line of interest and boxes represent region of interest (ROI) in BM and at bone surface. pH values extrapolated using ratio of HPTS 850/750 along the lines of interest with arrow presented in (E). Arrows 1–3 represent line of interest in BM and at bone surface based on their location. pH in different region of interest (ROI) including Cell-1, Cell-2, and bone marrow cavity (BC) in Fig 1E (scale bar = 50 μm, n = 3 biological replicates). Kinetics of pH change of IMDM medium up to 72 h incubation after initial adjustment of pH. Dashed and solid lines indicate calibration and experimental time window for pH ranges. Color of the medium precalibrated for different levels of pH at initiation of the culture (the 24 h time point in (H)) and after 40 h of culture further indicates pH stability. Download figure Download PowerPoint An ex vivo pH of 6.9 maintains HSC function To determine the effect of distinct levels of pH on the function of HSCs, 100 sorted LT-HSCs (Lin−Sca-1+c-Kit+CD34−Flt3− cells) from BM were exposed to a range of pH levels (6.4–7.8, Fig EV1H and I) at 3% oxygen for 2 days (40 h) and subsequently transplanted, alongside competitor cells, into primary recipient animals (Figs EV2A and 1B), and donor chimerism determined by flow cytometry (Figs 1B and EV2B). The level of chimerism in PB and BM to which donor HSCs contribute to in recipient animals in comparison to the competitor cells is an established measurement of HSC function. To determine the long-term engraftment and reconstitution potential following LT-HSC exposure ex vivo to pHs ranging from 6.9 to 7.8, BM cells from such primary mice were transplanted into secondary recipients (Fig EV2A), while animals receiving pH 7.4 HSC or pH 6.9 HSCs were subsequently followed in more detail, as exposure to pH 6.9 showed the highest levels of reconstitution of the pH 6.4–7.8 range tested in these experiments (Fig 1B). Noncultivated but also competitively transplanted LT-HSCs served as a reference in these experiments. LT-HSCs exposed to pH 6.9, in contrast to pH 7.4, conferred a higher level of chimerism in PB (significant) and BM (trend) in primary recipients (Fig 1C and D). The level of reconstitution driven by pH 6.9 HSCs in secondary recipients remained increased in PB as well as in BM (Fig 1F and H), now to a level that recipients from LT-HSCs cultured at pH 6.9 showed similar contribution compared to uncultured fresh LT-HSCs and significantly better than the contribution of pH 7.4 HSCs (Fig 1C, E, F and H). Animals reconstituted with HSCs exposed to a pH 6.9 showed no lineage bias and exhibited similar frequencies of T cells, B-cells, and myeloid cells in PB (Fig 1D and G) to that found in animals reconstituted by fresh, noncultured HSCs or HSCs exposed to a standard pH of 7.4. Distinct levels of pH, therefore, did not result in a different lineage differentiation potential of HSCs. Furthermore, percentages of HSC and progenitor cells (LT-HSCs, ST-HSCs, lymphoid-primed multipotential progenitor cells; LMPPs) within the primitive hematopoietic cell compartment were similar in primary recipient animals receiving pH 6.9, pH 7.4 or control HSCs (Fig 1I). There were only minor, but significant changes in secondary recipients, with the most prominent change being a reduced frequency of ST-HSCs in ex vivo cultivated HSCs, irrespective of whether exposed to pH 6.9 or 7.4 (Fig 1J). Exposure of HSCs to pH 6.9 did not differentially affect stem cell homing in a competitive stem cell homing assay (Fig EV2D), which excludes that the elevated level of chimerism in animals transplanted with pH 6.9 HSCs is linked to changes in homing of HSCs. Together, these results demonstrate that HSCs exposed to a pH of 6.9 for 2 days maintain their stem cell function in comparison to HSCs exposed to a pH of 7.4. Click here to expand this figure. Figure EV2. Analyses of HSC potential and homing Scheme of the experimental set-up for the competitive BM transplants. HSC (100 cells) from C57BL/6 mice (Ly5.2) were cultured under pH 7.4 and pH 6.9 settings for 40 h and transplanted with 1.5 × 105 cells from BoyJ mice (Ly5.1) under competitive settings into primary transplants, while for secondary transplants 2 × 106 BM cells from primary transplants transfer to irradiated BoyJ mice. Representative analysis of donor (Ly5.2) derived chimera and lineages (T, B, and myeloid cells) using flow cytometry. HSC (100 cells) from C57BL/6 mice (Ly5.2) were cultured under distinct pH (6.4–6.9) settings for 40 h and transplanted alongside 1.5 × 105 cells from BoyJ mice (Ly5.1). Bar graph represents contribution of total donor-derived Ly5.2+ cells in primary BM after 20 weeks in competitive transplants. n = 4 mice representative of two biological replicates. Schematic representation of the experimental setup for the competitive homing assay. Frequency of homed HSCs among total BM cells and frequency of homed pH 7.4 or pH 6.9 treated HSCs in total BM cells. n = 3 biological replicates, ns, non-significance using two-tailed unpaired Student's t-test. Experimental set-up for the competitive BM transplants. HSC (100 cells) from C57BL/6 mice (Ly5.2) were transplanted or cultured under pH 7.4 and pH 6.9 settings for 6 days and were then transplanted together with 1.5 × 105 cells from BoyJ mice (Ly5.1) under competitive settings into primary transplants, while for secondary transplants 2 × 106 BM cells from primary transplants were transplanted into irradiated BoyJ mice. Engraftment levels from untreated and cultured HSCs for 6 days at distinct levels of pH. Contribution of donor-derived Ly5.2+ cells to PB cells in primary and secondary recipients between 4 and 16 weeks post transplant. n = at least 9 mice per group from least three experiments (biological repeats). The bar graph represents contribution of donor-derived Ly5.2+ cells from untreated and 6 days cultured under pH 7.4 and pH 6.9 settings, data represent PB chimera in primary (upper panel) and secondary (lower panel) recipients at 16-week post transplant. n = at least 9 mice per group from least three experiments (biological repeats). **P < 0.01, ***P < 0.001, ****P < 0.0001 between marked groups. Statistical analysis using two-tailed unpaired Student's t-test. Data information: Data are means ± SE. Download figure Download PowerPoint We also tested the outcome of a longer term exposure (6 days) of HSCs to distinct levels of pH. HSCs kept for 6 days ex vivo at either pH 6.9 or pH 7.4 presented with a significant relative reduction in their ability to contribute to chimerism in primary and secondary recipients when compared to untreated (non-cultivated) HSCs, with HSCs exposed to a pH of 6.9 with an even lower level of chimerism than HSCs cultivated at pH 7.4 (Fig EV2E–G). The influence of changes in pH on HSC function upon longer term ex vivo culture is thus distinct from the maintenance effect of pH 6.9 with a short-term (40 h) exposure. To determine whether changes in pH also affect the function of hematopoietic progenitor cells (HPCs), cells from BM depleted for differentiated cells (LIN-cells) and thus enriched for progenitor cells were incubated in medium with a pH ranging from 6.9 to 8.0 for 2 days. Following incubation, the overall cell number and the HPC activity with a colony-forming cell assay were determined. While overall numbers were highest in cells exposed to a pH of 6.9 to 7.4, their numbers dropped dramatically at higher pH (7.6–8.0) levels (Fig EV3A). Furthermore, colony-forming unit cells, using a CFU-Cs assay, were decreased at a pH of 6.9, when compared to cells cultivated at pH 7.4 or higher (7.6–8.0; Fig EV3B). The data support that changes in extracellular pH also affect the function of HPCs. The full extent to which changes in pH will affect HPCs function will need to be further investigated. Click here to expand this figure. Figure EV3. Effects of pH on cell growth, CFUs, intracellular pH and size Total cell number recovered after cultivation of initial 4 × 105 Lin− cells under distinct levels of pH. n = 3 biological repeats. Representative images of colony formation ability at different pH condition and total numbers of CFUs present at 40 h after culture from initial 4 × 105 Lin− cells, Bar graph showing CFUs at pH conditions (pH 6.9–8.0). Data from n = 6 from three biological repeats, scale bar = 5 mm.**P < 0.01; using two-tailed unpaired Student's t-test. Gating strategy on flow cytometric data to identify stem cell enriched Lin- and LSK populations, HSC were identified as CD34− LSK cells for intracellular pH measurement. Representative flow cytometric analyses of fluorescence ratio of 640/580 nm (APC/PE channel) of the cell permeable pH sensitive probe carboxy SNARF-1 AM at pH 6.4, 7.2 and 8.0 that was used to derive pH calibration curve. pH calibration using bone marrow cells revealed a linear relationship (r2 = 0.95) between actual pH and measured ratios, in the pH range of 6.4–8.0, data represent n = 3 technical replicates. Intracellular pH (pHi) of HSCs and Lin− cells under extracellular pH 7.4 or pH 6.9 setting as measured by pH- Snarf-1 calibration curve in that population. n = 6 biological repeats, **P < 0.01, ***P < 0.001; using two-tailed unpaired Student's t-test. Representative flow cytometric overlay of FSC of HSCs cultured under pH 7.4 and pH 6.9 settings (representative of three biological repeats). Bar graph represents cell volume analysis using confocal images of HSCs cultured under pH 7.4 and pH 6.9 settings. Data are from > 80 cells per group and representative of three biological repeats **P < 0.01, ***P < 0.001; using two-tailed unpaired Student's t-test. Representative nuclear area of HSCs and quantification of HSCs cultured under pH 7.4 and pH 6.9 settings using confocal microscopy imaging. Scale bar = 3 μm. Data are from > 50 cells per group and representative of three biological repeats. **P < 0.01; using two-tailed unpaired Student's t-test. Representative flow cytometric overlay of CD34 expression on HSCs cultured under pH 7.4 and pH 6.9 settings. n = 3 biological repeats. Data information: Data are means ± SE. Download figure Download PowerPoint Extracellular pH 6.9 reduces growth, size, and metabolic stress of HSCs Changes in the extracellular pH (pHe) have been shown to modulate the intracellular pH (pHi; Casey et al, 2010; Damaghi et al, 2013). Intracellular pH was determined using carboxy SNARF-1 as a cell-permeable fluorescent pH probe, and pH values of HSCs were calculated based on a pH-SNARF-1 standard curve (Fig EV3C–E). These data demonstrated that a pHe of 6.9 led to a decrease in pHi of HSCs (Fig EV3F) and suggest that reduced pHi levels in HSCs may contribute to the functional changes in HSCs when exposed to a pHe of 6.9. Interestingly, a pHe of 6.9 also decreased the pHi in more differentiated progenitor cells (Lin negative cells) compared to the pHi following exposure to a pHe of 7.4 (Fig EV3F). To investigate likely mechanisms underlying the enhanced reconstitution potential of pH 6.9 HSCs, we determined the effect of pH 6.9 or 7.4 on HSC growth and proliferation. Culture of HSCs at pH 7.4 led to approximately 5-fold expansion of the cell number from HSCs, while a reduced but consistent 2-fold expansion at pH 6.9 was observed (Fig 2A). Changes in pHe have also been reported to alter cell size via altering the cytoskeleton (Busch et al, 1994; Kohler et al, 2012). While HSCs increased their size upon cultivation (Fig EV3G and H), HSC remained smaller when cultivated at pH 6.9 compared to 7.4, measured by regular microscopy, flow cytometry, and confocal imaging (Figs 2B, and EV3G and H), with a smaller nuclear volume (Figs EV3I and 4K). A smaller size of HSCs has been recently associated with an increase in stem cell potential (Lengefeld et al, 2021). Cell cycle analysis using standard DNA staining combined with EdU incorporation and flow cytometry implied a higher frequency of HSCs at G0/G1 phase and a lower number of HSCs entering S-phase at pH 6.9 in comparison to pH 7.4 (Fig 2C). A significantly increased frequency of cells positive for CD34, a marker associated with differentiation of murine HSCs, was observed at pH 7.4 compared to pH 6.9, which is consistent with enhanced maintenance of HSCs at pH 6.9 (Fig EV3J). Maintenance of HSC function at pH 6.9 thus correlates with a small ce" @default.
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• W4328049861 title "<scp>pH</scp> regulates hematopoietic stem cell potential via polyamines" @default.
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