Orbital shaking accelerates erythroid maturation
In static conditions, erythroblast (EBL) differentiation to enucleated reticulocytes is accompanied by a progressive loss of CD49d and increase in CD235a expression, taking approximately 10 days (Fig. 1A). We exposed EBL to orbital shaking to generate an average shear-stress of 1.4 Pa, comparable to the shear-stress at the point of the impeller of the bioreactors used4(estimation based on Odeleye22), but also to what can occur in the bone marrow microenvironment, as reviewed before23. Both static and dynamic cultures contained similar CD49d–/CD235a+ population at the end of the differentiation, however dynamic conditions showed significantly faster differentiation kinetics, reaching between 80%–90% CD49d–/CD235a+ cells 4 days earlier compared to static cultures (Fig. 1A, B). A significant increase in enucleated reticulocytes (DRAQ5– cells) was observed at day 4 and 6 in dynamic cultures compared to static cultures, which eventually progressed to similar enucleation percentages at the end of culture (Fig. 1C, D; Supplemental Fig. 1A, B)24. Faster differentiation was further confirmed by morphological analysis (Supplemental Fig. 1C). At the end of differentiation, dynamic cultures demonstrated a slight decrease – yet not significant – in EBL and reticulocytes production (Fig. 1E). Together these results indicate that orbital shaking leads to accelerated differentiation of EBL to enucleated reticulocytes.
Orbital shaker accelerates maturation and enucleation compared to static cultures. Erythroblasts (EBL) were differentiated for up to 10 days in dishes (static) or orbital shakers (dynamic). A) Erythroid cells were harvested at indicated days from dishes and flasks and stained with antiCD49d-PB (BD Biosciences, San Jose, CA, US ) (Alexa Fluor 405, y-axis) and antiCD235a-PE (OriGene Technologies, Rockville, MD, USA) (x-axis), and analysed by flow cytometry. B) Maturation states in static vs. dynamic conditions as explained in (A) were averaged for 5 donors (n = 5) by quantifying the percentage of cells within the respective gates Q1: CD49d+/CD235– immature EBL, Q2: CD49+/CD235a+ EBL, and Q3: CD49–/CD235a+ late EBL and reticulocytes. C) Enucleation was quantified by staining cells with the cell-permeable DNA-dye DRAQ5 (1 ng/mL, incubation 5 min at RT; BioLegend, San Diego, CA, USA). (APC, y-axis), forward scatter (FSC, x-axis) on the indicated days. Representative scatter plot displaying the gating for reticulocytes, EBL, and pyrenocytes is shown in Supplemental Fig. 1A. Enucleation percentage is defined as the %reticulocyte/ (%reticulocytes + %nucleated cells) using the gates as defined in Supplemental Fig. 1A. D) Enucleation states in static vs. dynamic conditions as explained in (C) were averaged for 5 donors (n = 5) by quantifying the percentage of cells within the respective gates: reticulocytes, erythroblasts, and pyrenocytes for 5 donors (n = 5). E) Cell-counts for reticulocytes and EBL that were differentiated in static or dynamic conditions for 4 donors (n = 4). The distinction between the two populations was done via DRAQ5 staining. B) 2way ANOVA test was performed, with ***p < 0.001, ** p < 0.01 and *p < 0.05. D and E) Paired two-tailed student t-test was performed, with p** p < 0.01 and *p < 0.05. Unless marked, no significance was observed.
Orbital shaking accelerates erythroblast differentiation immediately after induction
Next, we investigated whether dynamic conditions expedite differentiation at a specific maturation stage. Differentiating EBL were switched between static and dynamic conditions, as described in methods, at different time points and the enucleation rate was measured via flow cytometry. In line with Fig. 1, an inverse correlation between enucleated cells and the initial dynamic culture time was observed (Fig. 2).
Shear-induced enhanced maturation is progressively lost during erythroblast differentiation. A) Enucleation percentage (calculated as described for Fig. 1C) of samples switched from dynamic to static culture on Day 2, 4, or 6 of differentiation, or not switched, for 3 donors (n = 3). B) Enucleation percentage of samples switched from static culture to dynamic culture on Day 2, 4 or 6 of differentiation, as well as dish (static) and flask (dynamic) controls averaged for 3 donors (n = 3). Paired two-tailed student t-test was performed, with ** p < 0.01 and *p < 0.05. Unless marked, no significance was observed.
Cultures switched from orbital shaking to static conditions on day 2 showed slower enucleation kinetic compared to the samples switched on day 4. Importantly, all conditions reached comparable levels of enucleation indicating that the static cultures are not blocked in differentiation but that orbital shaking accelerates the process (Fig. 2A).
In the reverse experiment, transferring static cultures to dynamic conditions on day 2 and day 4 accelerated enucleation compared to the static control, but still lagged compared to cells under continuous orbital shaking. Switching at day 6 to orbital shaking conditions did not accelerate enucleation, suggesting that the regulation of accelerated differentiation occurs before day 6 (Fig. 2B). Together the data indicate that the orbital shaking induced accelerated differentiation occurs during the early phase of EBL maturation, between day 0 and 4.
Shear stress alters the erythroid transcriptome inducing HMGCR expression and cholesterol biosynthesis pathways
To start dissecting the underlying mechanism we performed transcriptomic analysis between static and dynamic conditions during the first 4 days of EBL differentiation.
Principal component analysis (PCA) demonstrated samples cluster per differentiation condition and time, where day 4 demonstrated the biggest difference (Supplemental Fig. 1D). Differential expression analysis between groups demonstrated many differentially expressed genes (DEGs) upon EBL differentiation, with 505 DEGs at day 4 of differentiation (Supplemental Fig. 1E, F; Supplemental table 1–3).
Pearson hierarchical and K-means clustering of these DEGs revealed 3 different gene expression profiles, assigned to 3 major clusters (Fig. 3A). Genes assigned to the first cluster (K1), showed an overall downregulation during differentiation in both static and dynamic cultures, but the downregulation levels were increased in dynamic conditions (Fig. 3A). The second cluster (K2) shows up-regulated genes in dynamic cultures; the third cluster (K3) represents up-regulated genes in static conditions.
RNA analysis between static and dynamic erythroblast differentiation confirms accelerated maturation and characterises the involved processes. Erythroblasts (EBL) of four different donors (n = 4) were differentiated for 4 days as indicated in material and methods. A) Heatmap showing a Pearson hierarchical clustering of z-scores of the 505 differentially expressed RNAs between Day1 and Day4. K-means clustering, reveals 3 clusters indicated as K1, K2, K3. Genes involved in the cholesterol biosynthesis pathway are reported next to their specific coordinates on the heatmap. B) The gene identifiers of the upregulated RNAs of K2 from (A) were extracted and their expression dynamics through complete differentiation of EBL to enucleated reticulocytes, as previously published by Heshusius24, was datamined and determined. Pearson hierarchical clustering was performed on z-scores over time in days as indicated (x-axis). Kmeans clustering (K = 3) was additionally performed and indicated as K2-a, K2-b and K2-c. C) ENRICHR analysis of K2-a to K2-c from (B) shows the top 10 enriched Gene Ontology (GO) term biological processes that are associated with the RNAs in the indicated Kmeans cluster according to the adjusted p-value. D) STRING analysis of the genes within K2-b was performed and genes within the first 3 biological processes according to the false discovery rate highlighted in red, blue, and green. Lines thickness indicates the strength of data support. E) Representation of cholesterol biosynthesis pathway with related z-score of genes up-regulated on day 4 in dynamic (D) compared to static (S) conditions. F) HMGCR (ab242315, Abcam) expression was assessed through western blot analysis, during EBL differentiation of 3 different donors (n = 3) in static and dynamic conditions. GAPDH (MAB374, Millipore) was used as loading control. G) Quantified expression of HMGCR during cell differentiation in dynamic and static conditions. HMGCR expression was normalised according to GAPDH expression. G) 2way ANOVA test was performed, with ** p < 0.01 and *p < 0.05. Unless marked, no significance was observed.
Gene ontology analysis revealed that genes in K1 and K3 are enriched for DNA replication and nucleosome organisation processes, respectively (Supplemental Fig. 2A). Moreover, K3 genes during differentiation revealed a cluster of genes upregulated during differentiation (Supplemental Fig. 2C). The differentiation-associated gene expression profile in K1 and K3 is in line with previously reported dataset showing similar gene expression dynamics during differentiation to enucleated reticulocytes (Supplemental Fig. 2B and C24).
In contrast, transcripts within K2 were down-regulated on day 4 in static conditions but up-regulated in dynamic environments. To assess the expression of these genes throughout the complete differentiation to reticulocytes, we compared our data more closely to those from Heshusius24 and re-clustered them based on their gene expression profile, giving rise to 3 additional clusters (K2-a/b/c, Fig. 3B).
The top 10 regulated biological processes according to the adjusted p-value (EnRicHR) revealed that genes in K2-a regulate response to heat and to unfolded protein and chaperone cofactor- dependent protein refolding and are upregulated starting from ~ day 7 of differentiation in static condition. Genes within K2-c are involved in the regulation of cell migration and response to ER stress and are upregulated earlier in differentiation, between day 5 and 6. In contrast, K2-b demonstrated a strong enrichment for genes mostly involved in cholesterol/steroid metabolism being up-regulated in dynamic conditions but downregulated during differentiation in static coltures (Fig. 3C). K2-b STRING analysis confirmed the presence of a cluster of genes involved in secondary alcohol metabolic and cholesterol biosynthetic processes, among which also HMGCR, a rate limiting enzyme of the cholesterol biosynthesis pathway and fundamental coordinator of mevalonate metabolism that assures constant de novo synthesised cholesterol and non-sterol products (Fig. 3D)25. Within the cholesterol biosynthesis pathway, the enzymes HMGCS1 and HMGCR, IDI1, SQLE and HSD17B7 showed a similar trend, being upregulated in dynamic conditions (Fig. 3E). Of note, the protein expression of HMGCR peaked at day 4 and 5 of differentiation, where peak expression during dynamic conditions occurs during day 1 and 2 and decreased on day 3 and 4 (Fig. 3F and Supplemental Fig. 2D), providing further proof of the accelerated maturation in dynamic cultures. Importantly, the high expression of HMGCR detected on day 1 and day 2 of differentiation in dynamic conditions was never reached in static cultures, suggesting an adaptation response of the cells to the shear-stress, and validating the increase in gene expression observed by RNA-sequencing (Fig. 3G). In all, these data confirm accelerated differentiation of EBL upon dynamic conditions and provide a transcriptional and possible functional footprint of downstream effects of shear-induced cholesterol transcriptional program.
Lipidomic analysis of erythroblasts confirms a differential lipid composition between dynamic and static conditions
To assess the effect of the upregulated lipid transcriptional program on day 4 of erythroid differentiation, EBL were subjected to mass spectrometry-based lipidomics analysis. Partial least squares-discriminant analysis (PLS-DA) demonstrated that samples cluster per differentiation condition (Fig. 4A). Clustering of the top 75 lipids selected by adjusted p-value shows that at day 4 of differentiation, static cultures have higher levels of long chain fatty acids and numerous phospholipids (Fig. 4B, cluster A), while dynamic cultures are marked by elevated levels of cholesteryl esters (ChE), free cholesterol (Ch), triglycerides (TG), diglycerides (DG), and phosphatidylcholines (PC) (Fig. 4B, cluster B). Assessment of the contributions of different lipid classes shows that dynamic culturing enriches the EBL lipidome in triglycerides (TG 9%) cholesterol (Ch 16%) at the expense of phosphatidylethanolamine (PE, 7%), fatty acids (FA, 5%) and phosphatidylcholine (PC, 46%), while static cultures show increased percentages of phosphatidylcholine (PC, 51%) phosphatidylethanolamine (PE, 11%) and fatty acids (FA, 11%) (Fig. 4C—outlined in yellow and Fig. 4D). These findings corroborate the upregulation of genes involved in the cholesterol and lipid biosynthesis previously described in dynamic cultures.
Day 4 erythroblasts differentiated in dynamic conditions show higher concentration of cholesterol triglycerides and phosphatidylcholine compared to cells differentiated in static. Day 4 erythroblast (EBL) of three different donors (n = 3) were characterized by global lipidomics to determine their lipid composition. A) Partial Least Squares-Discriminant Analysis (PLS-DA) of samples differentiated in static and dynamic conditions. X-axis represents variations between conditions, y-axis represents variations between donors. B) Heatmap of the top 75 lipids differentially expressed at day 4 of differentiation in static (left- red) and dynamic (right- green) conditions according to the adjusted p-value. Cluster A represents lipids upregulated in static and cluster B lipids upregulated in dynamic. C) Pie-charts representing the lipid composition in percentage of day 4 EBL obtained from static and dynamic cultures. The most expressed lipids in static and dynamic condition are outlined in yellow. D) Comparison of the single lipids detected in reticulocytes obtained from static and dynamic conditions. X-axis represents the abbreviation of lipids name, y-axis the area under the curve. Paired two-tailed student t-test was performed, with ** p < 0.01 and *p < 0.05. Unless marked, no significance was observed.
Reticulocytes generated in shear-stress environment show higher cell stability, but similar lipid composition compared to reticulocytes generated in static conditions
We next investigated whether shear-stress induced transcriptional program can affect erythrocyte homeostasis and reticulocyte plasma membrane stability and deformability (Fig. 5).
Reticulocytes obtained from dynamic cultures show higher cell stability but similar deformability and lipid composition when compared to static. Erythroblasts (EBL) of three different donors (n = 3) were differentiated for 12 days as indicated in material and methods. On day 12 of differentiation, cells were filtered, and the obtained reticulocytes subjected to cell deformability and stability assays. Measure of the reticulocyte’s deformability was detected using the ARCA. Cells were subjected to a shear stress of 3 Pa and 10 Pa. A schematic representation of the ARCA is shown in supplemental Fig. 4D. A) The x-axis represents the ratio between the length and the width of the cell (A/B), the y-axis is the normalised number of cells. A/B ratio directly correlates with reticulocyte deformability. B) Hemolysis percentage (y-axis) measured after incubation with different concentrations of NaCl solution (x-axis) was assessed to determine the stability of reticulocytes obtained from static and dynamic cultures. A and B) Native RBCs were used as control in deformability and stability assays. A) Unpaired two-tailed student t-test of the area under the curve of static condition vs RBC and dynamic condition vs RBC was performed, with *** p < 0.001, ** p < 0.01 and *p < 0.05. B) 2way ANOVA test was performed, with*** p < 0.001, ** p < 0.01 and *p < 0.05. Unless marked, no significance was observed.
We measured the deformability of the cultured reticulocytes using an automated rheoscope and cell analyzer (ARCA) subjecting the cells to a shear-stress of 3 and 10 Pa (Supplemental Fig. 4D). We detected lower, but yet not significant, deformability in reticulocytes obtained from static and dynamic cultures compared to native RBC, no significant difference was detected between static and dynamic conditions (Fig. 5A). In contrast, the osmotic resistance of reticulocytes derived from dynamic cultures was comparable to RBC controls where static cultures showed significant lower osmotic resistance (Fig. 5B).
Interestingly, global lipidomics analysis of reticulocytes showed minimal to no differences between static and dynamic cultures, aside from a slight increase in fatty acids in static cultures (Supplemental Fig. 3). The data suggest that the transcriptional changes detected at the onset of EBL differentiation in dynamic conditions, is associated with higher osmotic resistance of reticulocytes generated in shaker, but similar lipid composition compared to static conditions.
The HMGCR inhibitor lovastatin leads to a failure in adaptation to orbital shaking conditions and a severe reduction of reticulocyte formation during erythroblast differentiation
Increased production of cholesterol in dynamic conditions poses the question whether the increased HMGCR expression is essential for the adaptive response of differentiating EBL during dynamic conditions. Statins are well-described HMGCR inhibitors used in the clinic to reduce cholesterol levels26,27.
Lovastatin treatment at different concentrations did not affect cell yield and general hemoglobinization during EBL differentiation in static cultures, with the exception of 1 µM treatment. In contrast, dynamic cultures treated with the same lovastatin concentrations showed a significant reduction of cell viability and hemoglobinization (Fig. 6A, C, E and Supplemental Fig. 4). CD49d/CD235a marker expression was not affected in lovastatin-treated EBL differentiated in static conditions, except for a reduction of CD235a+/CD49d– cells with 1 µM lovastatin concentration (Fig. 6B, D). We were unable to characterize differentiation markers for dynamic cultures in presence of lovastatin because most of the cells did not survive the HMGCR inhibition (Fig. 6B, D, E).
Lovastatin inhibition of HMGCR prevents cells adaptation to shear stress environment. Erythroblasts (EBL) of three different donors (n = 3) were incubated with different concentrations of lovastatin on day 0 of differentiation and cultured in static and dynamic conditions. Erythroid cells were harvested on day 12 of differentiation and analysed by flow cytometry. A) Representative example of living gate strategy plotting forward scatter (FSC x-axis) vs side scatter (SSC y-axis) in cells differentiated in static and dynamic conditions treated with different concentrations of lovastatin. B) Maturation states of cells differentiated in static and dynamic conditions incubated with different concentrations of lovastatin according to antiCD49d-PB (BD Biosciences, San Jose, CA, US) (Alexa Fluor 405, y-axis) and antiCD235a-PE (OriGene Technologies, Rockville, MD, USA) (x-axis), staining. C) Quantified percentage of cells in the living gate, averaged for 3 donors (n = 3). Gating strategy to identify the living cells gate is described in Supplemental Fig. 4). D) Quantified maturation states of cells differentiate in static and dynamic incubated with different concentrations of lovastatin were measured as described in Fig. 1A. Percentage of cells in gates was averaged for 3 donors (n = 3). ND indicates not-detectable measurement due to the low percentage of cells in the living gate. E) Representative picture of pelleted cells on day 12 of differentiation incubated with different concentration of lovastatin. C and D) 2way Anova test was performed, with *** p < 0.001 ** p < 0.01 and *p < 0.05. Unless marked, no significance was observed.
Overall, the data shows that HMGCR enzymatic inhibition was not tolerated during EBL differentiation under turbulent culture conditions and thus it is required for the adaptive response of EBL to orbital shaking.
S1P inhibition prevents SREBP cleavage and activation reducing HMGCR expression and inducing cell death in dynamic-differentiated cells
SREBPs (Sterol-regulatory- element-binding-proteins) are master regulators of cholesterol metabolism in many cells. The precursor of SREBPs is located in the ER and when the cellular steroids level is reduced, a cascade of cleavage processes, initiated by the site-1 protease (S1P), allows SREBPs translocation into the nucleus, where they regulate the expression of genes involved in cholesterol metabolism, such as HMGCR28. To explore if the increase of HMGCR expression detected during erythroid differentiation in dynamic conditions, was due to the cleavage and subsequent activation of SREBPs, we inhibited S1P via the administration of its inhibitor, PF-429242 (1 µM). PF-429242 effects were evaluated 24 and 48 h after its administration (Fig. 7). PF-429249 did not alter the total cell count on day 1 of differentiation, with a slight reduction of EBL differentiated in static conditions on day 2. Of note, the cell count of EBL differentiated in dynamic environment was similar between the untreated-controls and the samples subjected to S1P inhibition, during the 2 days of erythroid maturation (Fig. 7A). The reduction in hemoglobinization after PF-429242 treatment suggests an increase in cell death in dynamically differentiated cells after S1P inhibition (Fig. 7B). In line with this, flow cytometry data showed that the inhibition of S1P induced a reduction of cells in the living gate in samples differentiated in dynamic condition on day 1. The reduction in living cells was also detected in static cultures on day 2, but it did not reach the levels observed in dynamic conditions on the same day (Fig. 7C, D and Supplemental Fig. 4). Moreover, an increase of more mature cells, CD235a+/CD49d–, was observed on day 1 and 2, in both static and dynamic cultures, with a stronger effect in the latter one, reaching ~ 80% of CD235a+/CD49– EBL on day 2, possibly related to a selection of the most mature cells after the S1P inhibition (Fig. 7E). HMGCR expression, evaluated by Western blot, was severely reduced in both static and dynamic cultures treated with PF-429242 on day 1 and 2 of differentiation. BAND 3 was used as loading control. (Fig. 7F, G and Supplemental Fig. 5). All together this data suggests a novel role for the S1P-induced cleavage of SREBPs to transcriptionally induce HMGCR expression and increase cholesterol biosynthesis in dynamically differentiated EBL.
S1P inhibition in the first 2 days of differentiation, reduced cell viability and HMGCR expression and led to a loss of EBL viability in dynamic cultures. Erythroblasts (EBL) of three different donors (n = 3) were incubate with the S1P inhibitor PF-429242 on day 0 of differentiation and cultured in static and dynamic conditions for 2 days. The effects of S1P inhibition were evaluated every 24 h. A) Cell-counts for EBL that were cultured in static or dynamic conditions in presence or absence of the inhibitor. B) Representative picture of pelleted cells of 3 donors (n = 3) on day 2 of differentiation in static or dynamic conditions, incubated with PF-429242. C) Representative example of living gate strategy plotting forward scatter (FSC x-axis) vs side scatter (SSC y-axis) in cells differentiated in static and dynamic conditions treated with PF-429242. D) Quantified percentage of cells in the living gate, averaged for 3 donors (n = 3). Gating strategy to identify the living cells gate is described in Supplemental Fig. 4. E) Maturation states in static vs. dynamic conditions as explained in Fig. 1A were averaged for 3 donors (n = 3) by quantifying the percentage of cells within the respective gates Q1: CD49d–/CD235a–, Q2: CD49d+/CD235– immature EBL, Q3: CD49–/CD235a+ late EBL and reticulocytes, Q4 CD49+/CD235a+ EBL. Representative example of gating strategy corresponding to the described conditions are reported below. F) HMGCR expression assessed through western blot analysis, during 2 days of EBL differentiation in static and dynamic conditions treated with PF-429242 and controls. BAND 3 was used as loading control. G) Quantified expression of HMGCR during cell differentiation in dynamic and static conditions. HMGCR expression was normalised according to BAND3 expression. (A, D, and G 2way Anova test was performed, with **** p < 0.0001, *** p < 0.001 **, p < 0.01 and *p < 0.05. Unless marked, no significance was observed.
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- Source: https://www.nature.com/articles/s41598-024-81746-8