Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model

<div><p>Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as ‘the storage lesion’. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34⁺ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated <i>ex vivo</i> stemRBCs from primary human cord blood CD34⁺ cells and compared them to donor-derived RBCs based on a number of <i>in vitro</i> parameters. <i>In vivo</i>, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.</p></div

Proteomic analysis of hemoglobin expression.

<p>(A) Ratio of spectra (%) matched specifically to Hb tryptic peptides (spectral count) over total matched spectra during erythropoietic differentiation of cord blood-derived CD34⁺ cells along with human control RBCs and filtered cells on day 18. (B) Intensity based absolute quantification (iBAQ) was used to calculate the mole % ratio of peptides unique to α, β and γ Hb subunits over all identified peptides of filtered (F) and non-filtered (NF) cells on day 18 of culture along with human control RBCs. Other Hb subunits were found but were <0.5% of total Hb and are thus not shown on the graph. Standard deviations for α, β, γ, and δ are as follows: (NF): 3.4%, 2.8%, 3.0%, and 0.05%; (F): 4.2%, 9.7%, 5.8, and 0.23%; (control) 6.8%, 7.0%, 0.05%, and 0.36%, respectively.</p

Characterization of stemRBCs.

<p>(A) Cell diameters of stemRBCs (n = 10) derived from cord blood isolated CD34⁺ cells during differentiation from day 0 to day 18 in culture compared to human control RBCs and filtered cells on day 18. (B) Cell morphology of adult human blood (control) from cytospins for comparison with pre- (NF) and post-filter (F) day 18 stemRBCs. Scale bar represents 20 μm. (C) Total number of viable, non-adherent cells were determined and plotted as a mean ± SE of 4 cultures. (D) Frequency of cells expressing several erythroid markers as determined by flow cytometry over an 18 day culture period (mean ± SE) and (E) mean ± SE fluorescence intensities (MFIs) showing expression patterns of these markers during differentiation (n = 8).</p

Functional analysis of stemRBCs <i>in vivo</i>.

<p>(A) In vivo recovery of day 18 stemRBCs and WT mouse control RBCs (n = 4–6 per group) in anemic SCID mice. CFSE-labeled cells were infused by i.v. injection (tail vein). Mouse whole blood was collected at the indicated times and analyzed by flow cytometry. Recovery was set to 100% at the 5 min time-point. (B) Representative images of mouse whole blood collected at 5 minutes post-injection of murine control RBCs or stemRBCs (arrows). Scale bar: 20 μm. (C) The animal model of oxygen function was validated by subjecting anemic C57BL/6J (Hbb^th3/+) and wildtype C57BL/6J mice (n = 3 per group) to a 15 min swim (shown as an orange rectangle) in 26°C water and ambient air. Lactate levels were measured pre- and post-swim, and every 15 min thereafter. Oxygen deficit due to exercise is reported in mice through a corresponding spike in lactate level. (D) stemRBCs, along with controls (saline and wild-type mouse RBCs; n = 3–5 per group) were introduced into C57BL/6J Hbb^th3/+ mice. After 30 min, lactate was measured, and mice were subjected to a 15 min swim. Lactate was measured post-swim and every 15 min as indicated. Data points are means and error bars show SEM. (* = p< 0.05).</p

Fold change comparison of hemoglobin subunits between stemRBC and control RBC.

<p>Non-filtered and filtered stemRBC on day 18 of culture along with human control RBCs. Mean, (n = 3).</p

A Coordinated Proteomic Approach for Identifying Proteins that Interact with the <i>E. coli</i> Ribosomal Protein S12

The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions (Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of <i>Escherichia coli</i> ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface

A Coordinated Proteomic Approach for Identifying Proteins that Interact with the <i>E. coli</i> Ribosomal Protein S12

The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions (Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of <i>Escherichia coli</i> ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface

A Coordinated Proteomic Approach for Identifying Proteins that Interact with the <i>E. coli</i> Ribosomal Protein S12

The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions (Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of <i>Escherichia coli</i> ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface

A Coordinated Proteomic Approach for Identifying Proteins that Interact with the <i>E. coli</i> Ribosomal Protein S12

The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions (Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of <i>Escherichia coli</i> ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface

A Coordinated Proteomic Approach for Identifying Proteins that Interact with the <i>E. coli</i> Ribosomal Protein S12

The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions (Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of <i>Escherichia coli</i> ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface