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S. Bhatlekar et al.
platelet disorders. Improvements in this technology may also allow 'designer' platelets to be engineered that over- come immunological incompatibility and infection issues.5
A major limitation of all MK culture systems is the rela- tively short time period for which the differentiating MK can be kept viable before they die through apoptosis.6 The role of apoptosis during MKpoiesis is somewhat con- troversial,7 with some data supporting a role for the intrin- sic pathway of apoptosis in platelet production,8-10 while other studies show that MK must restrain apoptosis to survive and progress safely through proplatelet formation and platelet generation.11-13 However, the apoptosis regula- tors of human cord blood-derived (CB)-MK cultures remain poorly understood. During the course of our stud- ies with cultured CB MK, we observed two distinct popu- lations of cells by forward and side scatter flow cytometry. The aims of the current study were to determine: (i) whether there were viability and apoptosis differences between these two populations; and (ii) molecular mech- anism(s) of apoptosis regulation during Mkpoiesis. We also wanted to begin to identify approaches that would reduce MK apoptosis in order to produce greater yields of MK and platelet in cultures. We demonstrate that the anti-apoptosis Bcl2 family member BCL2L2 (encoding Bcl- w) regulates cultured MK apoptosis, promotes proplatelet formation, and is associated with human platelet number.
Methods
Primary megakaryocyte cultures
Human umbilical cord CB was obtained from the New York Blood Center (New York, NY, USA) under institutional review board (IRB) approval (00108527). CD34+ hematopoietic stem and progenitor cells (HSPC) were isolated from human umbilical vein CB and cultured in serum free expansion media supplemented with 25 ng/mL of stem cell factor (SCF) and 20 ng/mL of throm- bopoietin (TPO) (Peprotech, Rocky Hill, NJ, USA) for six days. Cells were cultured with 50 ng/mL TPO only from days 6-13.14 Adult granulocyte-macrophage colony-stimulating factor (GM- CSF) mobilized CD34+ HSPC were purchased from the Utah Cell Therapy and Regenerative Medicine Center (Salt Lake City, UT, USA) under the University of Utah IRB approval (00108527).
Megakaryocyte proplatelet formation assay
Day 9 transduced cells were plated at 2x104 cells/mL in 60μ-Dish Grid-500 plates (Ibidi, Fitchburg, WI, USA) using fresh medium supplemented with 50 ng/mL TPO. On day 13, the pro- platelet forming (PPF) MK, defined as displaying at least one fila- mentous pseudopod, were scored with a light microscope blinded as to experimental group. Images were taken at room tempera- ture under 40x objective, numerical aperture 1.35, using FV1000 confocal laser scanning microscope (Olympus, Center Valley, PA, USA). The percentage of PPF MK was calculated as the number of PPF MK compared to the total number of round cultured cells ana- lyzed. An average 200 cells were counted per condition.
Integrin αIIbβ3 activation assessment
Cells were resuspended in Tyrode’s buffer (138 mM NaCl, 5.5
mM dextrose, 12 mM NaHCO3, 0.8 mM CaCl2, 0.4 mM MgCl2, 2.9 mM KCl2, 0.36 mM Na2HPO4, 20 mM Hepes, pH 7.4). Integrin αIIbβ3 activation was quantified with FITC-labeled PAC1 (1:100) (BD Pharmingen) in response to stimulation15 with no agonist (resting), 100 μM PAR4-AP [GL Biochem (Shanghai) Ltd., China], 250 nM thrombin (Enzyme Research, South Bend,
IN, USA) or 10 μg/mL CRP (synthesized at Baylor College of Medicine and cross-linked with glutaraldehyde) for 20 minutes (min) at 37°C, followed by 4% paraformaldehyde fixation at room temperature. Cells were analyzed on a Cytoflex or BD Accuri C6 flow cytometer.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 6 software version 10.1 (La Jolla, CA, USA) and reported as Mean±Standard Error of Mean (SEM). Fold changes for BCL2L2 levels over time in cultures and for lentiviral overexpression were presented as log2 (fold change) compared to their respective con- trols (day 6 for changes in BCL2L2 levels over time and empty vec- tor control for overexpression) and analyzed by a one-sample t- test. Log2 transformation was adopted to have a normally distrib- uted fold-change data, determined by Kolmogorov-Smirnov nor- mality test. The relationship between platelet BCL2L2 mRNA expression levels and platelet counts in the 154 healthy human donors in the Platelet RNA Expression Study 1 (PRAX-1) was assessed by Pearson’s correlation with 95% Confidence Interval (95%CI). For all other analyses, statistical significance was assessed using paired Student t-test. P<0.05 was considered statis- tically significant.
See Online Supplementary Appendix for further details of the experiments.
Results
Subsets of cultured megakaryocytes show differing viability
CD34+ cells were isolated from human umbilical vein CB and cultured to generate MK as described previously.14 By day 13, CD34+ cells were differentiated into MK, as demonstrated by the expression of mature MK markers (Online Supplementary Figure S1A-D). Day 13 MK were larger than day 0 HSC (Online Supplementary Figure S1E) and demonstrated polyploid MK (Online Supplementary Figure S1F and G). Thus, these culture conditions promot- ed generation of mature, polyploid MK.
Flow cytometric analysis of day 13 cultures revealed dis-
tinct cell populations differing by forward scatter (FSC)
and side scatter (SSC) characteristics (Figure 1A): larger
MK with lower granularity [larger, lower granular (LLG)]
and smaller MK with higher granularity [smaller, higher
granular (SHG)]. Peripheral blood (PB) mobilized adult
CD34+ cells cultured under the same conditions also gen-
erated similar subpopulations of MK (Online Supplementary
Figure S2). Approximately 80% of LLG MK expressed
both CD41a/CD42a and CD41a/CD42b markers, while
only 20-30% SHG MK were double positive for mature
MK markers (Figure 1B). Lower surface expression of
CD42b (GPIbα) in day 13 SHG cells compared to LLG
cells is consistent with mitochondrial damage16 as
observed in apoptotic adult mobilized HSPC-derived MK.17
Since the numbers of SHG MK showed less differentia- tion towards mature MK than the LLG MK, and because SHG MK showed reduced CD42b expression, we consid- ered whether SHG MK may have undergone (or be under- going) apoptosis. Figure 1C shows few CD41a+ LLG MK- bound annexin V [termed phosphatidylserine (PS)Low], a marker of apoptosis-induced phosphatidylserine expres- sion, whereas a significantly higher percentage of CD41a+ SHG MK bound annexin V (termed PSHigh). Anti-annexin
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haematologica | 2019; 104(10)