Erum Ahmed_mbp Final Report

Erum Ahmed

MBP Final Report Abstract Introduction Platelet transfusions are required for many different thrombotic deficiencies, resulting from chemotherapy or blood disorders. Donated platelets carry many risks that could be eliminated by producing autologous or compatible platelets from megakaryocytic cells grown in culture from a patient’s own hematopoietic stem cells1. In order to increase the number of platelets that can be produced from cultured stem cells, megakaryocytic ploidy and proplatelet formation must be increased. It has been found that nicotinamide (NIC), or vitamin B3, is very effective in increasing megakaryocytic ploidy and proplatelet formation2. The goal is to generate enough platelets for one transfusion from one million CD34+ cells, or 500,000 platelets per CD34+ cell1. Platelets and megakaryocytes originate from the myeloid lineage of hematopoietic stem cells, as shown in Figure 1.

Figure 1. Eight major hematopoietic lineages arise from self-renewing multipotent stem cells.

Specifically, this study aims to design culture conditions to promote megakaryocyte differentiation into proplatelets, and eventually platelets. Tissue culture surfaces coated with fibrinogen, fibronectin, vitronectin, and type I collagen were used in this study to evaluate their effect on proplatelet formation in the human CHRF cell line and in primary human and murine megakaryocytes. Past studies showed that primary megakaryocytes from murine bone marrow

aspirates grown on various protein surfaces showed the highest degree of proplatelet formation when cultured on fibrinogen3.

Figure 2. Inhibition of α IIbβ 3 prevents proplatelet formation on fibrinogen, vitronectin, and VWF/botrocetin, but not laminin. Bone marrow aspirates from Tpo-treated mice were plated on these proteins with and without α IIbβ 3 inhibitors. Proplatelet formation on fibrinogen is more extensive than on the other proteins in the presence of α IIbβ 3.

Vitronectin and fibronectin also promote adhesion and were investigated as surface coatings to promote proplatelet formation3,4. Recent cultures of primary human Mks have not shown proplatelet formation to the degree that had been seen in previous cultures done in this laboratory, so the goal of this study is to promote greater proplatelet formation through the use of ECM protein coated surfaces. CHRF cells were cultured with and without PMA, which induces differentiation, and primary megakaryocytes were cultured on protein-coated surfaces with thrombopoietin (Tpo) to induce differentiation. Various cytokine cocktails were also tested to assess their effect on proplatelet formation. Cells were treated with NIC at different time points to further promote proplatelet formation. Proplatelet formation was evaluated by observation after staining the cells. Some work was also done in continuation of a project by Ana Sofia Garcia involving the development of ligand-presenting surfaces. The basis of this project is the strong adhesive attachment found in the attachment pads of marine mussels. Foot proteins found in these pads closely resemble the dopamine molecule5. Drs. Haeshin Lee and Phil Messersmith developed a method of using polydopamine to coat surfaces, with the advantage being that any molecules with free amine or thiol groups can be covalently bound to the polydopamine layer for ligand presentation6. Specifically, the protein NeutrAvidin, a deglycosylated version of avidin, was covalently bound to the polydopamine (pDA). NeutrAvidin’s strong affinity for biotin allows it to bind biotinylated peptides for ligand presentation. This could eventually be developed into a culture system where cells would bind and adhere strongly to peptides presented in this way 5. In this study, the amount of NeutrAvidin bound to pDA-coated substrates was quantified using a protein assay. This was done to ensure that the current coating protocols were allowing enough NeutrAvidin to bind such that the pDA surfaces were being functionalized by the addition of the protein. Materials & Methods ECM Proteins

Fibrinogen (Innovative Research), fibronectin (Sigma), vitronectin (Sigma), and Type I collagen (Sigma) were coated onto 6-well tissue culture well plates (Becton Dickinson) based on protocols supplied by the protein vendors. Fibrinogen was coated at 1 μg/cm2, fibronectin was coated at 3 μg/cm2, and vitronectin was coated at 0.1 μg/cm2. Plates were stored at 4°C until use and equilibrated to 37°C prior to the addition of cells and media. CHRF Cell Line CHRF cells were cultured in IMDM + 10% FBS, and 6-well plates were coated with fibrinogen, fibronectin, vitronectin, or Type I collagen. The cells were seeded onto the protein-coated wells at 75,000 cells/mL, with and without PMA added. The cells were stained with Wright-Giemsa (Camco) at Day 8 and imaged at 40x magnification. Polystyrene controls, both untreated and PMA-treated, were also seeded and stained. Primary Murine Megakaryocytes Primary murine megakaryocytes were isolated from mouse bone marrow aspirates and cultured in DMEM + 10% FBS + 1% pen/strep. Media was supplemented with rhTpo, rhSCF (recombinant human stem cell factor), and rmIL-3 (recombinant murine interleukin-3). 6-well plates were coated with fibrinogen, fibronectin, and vitronectin. Cells were seeded onto the protein-coated wells at 1 x 106 cells/mL. 12.5 mM NIC was added to half the wells at Day 1 after seeding. The cells were stained with Wright-Giemsa at Day 4, and the fixed and stained cells were imaged with a Leica microscope at 40x magnification. Polystyrene controls, +/- NIC treatment were also seeded and stained. Primary Human Megakaryocytes Primary human megakaryocytes were cultured in X-VIVO 20 supplemented with varying cytokines depending on the experiment. Dead cell removal was performed in some cultures and cells were grown in T-flasks at 5% O2 and pH 7.2 for 5 days. Cells were seeded onto ECMprotein coated wells or coverslips at either Day 5, Day 8, or Day 9, at which point they were treated with NIC. Wright-Giemsa staining was done at an appropriate timepoint to visualize proplatelet formation. 

Trial 1: Primary human Mks cultured in X-VIVO 20 suppledmented with 100 ng/mL Tpo, 100 ng/mL SCF, 10 ng/mL IL-11, 10 ng/mL IL-6, and 25 U/mL heparin were seeded at 100,000 cells/mL on Day 13 on ECM protein-coated surfaces. Half of the wells were treated with 6.25 mM NIC. By Day 20, proplatelet formation was still not visible. At this point, a co-stain was performed for CD41 and CD62p on the flow cytometer, to determine the maturity of the cells and whether any platelets had formed. 1 mL of suspended cells from each well was taken for this stain and the cultures continued with the remaining media. Some proplatelet formation began around Day 24, and at Day 28 of the culture, WrightGiemsa staining was done to capture the proplatelet extensions. A few long extensions were present in fibrinogen- and fibronectin-coated wells, without NIC treatment. At this late timepoint, NIC treatment had no effect and may have been inhibitory.

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Trial 2: Primary human Mks cultured in X-VIVO 20 supplemented with 100 ng/mL Tpo only were seeded at 80,000 cells/mL on Day 7 on fibrinogen, fibronectin, and uncoated polystyrene surfaces, treating half of the wells with 6.25 mM NIC. Dead cell removal had

been performed on Day 2, to improve cell growth and maturation. Proplatelets began forming very quickly, and a few were visible already after one day. At Day 11, proplatelet formation was not much different than it had been at Day 8. On Day 12, which was 5 days after NIC treatment, the cells were stained with Wright-Giemsa. 

Trial 3: Primary human Mks were cultured in X-VIVO 20 supplemented with 100 ng/mL Tpo, 100 ng/mL SCF, 10 ng/mL IL-6, 10 ng/mL IL-11, and 25 U/mL heparin. At Day 12, cells were seeded at 80,000 cells/mL into fibrinogen-coated wells and polystyrene wells, with half the wells receiving 6.25 mM NIC. 2 mL of culture was added to each well.

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Trial 4: Primary Mks were thawed and seeded into 3 flasks at Day 0:

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T-150 flask containing 3,122,760 cells in 40 mL of X-VIVO 20 plus 100 ng/mL Tpo

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T-25 flask containing 304,000 cells in 6 mL of X-VIVO 20 plus 100 ng/mL Tpo, 100 ng/mL SCF, 10 ng/mL IL-11, 10 ng/mL IL-3

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T-25 flask containing 522,000 cells in 10.44 mL of X-VIVO 20 plus 100 ng/mL Tpo, 100 ng/mL SCF, 10 ng/mL IL-11, 10 ng/mL IL-6 Cell numbers were chosen based on cell expansion predictions. These grew in 5% O2 and pH 7.2 until Day 5, when dead cell removal was performed and the flasks were switched to 20% O2 and pH 7.4. At this point, one set of uncoated coverslips (NUNC™ Brand Thernanox) and fibrinogen- and fibronectin-coated coverslips were seeded with 100,000 cells/mL. The rest of the coverslips were seeded at Day 8. The cells were observed very closely after seeding onto coverslips to watch for proplatelet formation. They were stained with Wright-Giemsa as well as β-tubulin fluorescence staining at Day 12.

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Trial 5: Primary Mks grew in 5% oxygen and pH 7.2 with 100 ng/mL Tpo & SCF + 10 ng/mL IL-11 & IL-6 + 2.5 ng/mL IL-3. On Day 5, dead cell removal was performed on these cells and they were separated into two flasks, one with the same cytokines they had been growing in, and one with 100 ng/mL Tpo, 100 ng/mL SCF, 10 ng/mL IL-11, 10 ng/mL IL-3. The flasks were kept at 20% oxygen and pH 7.4. On Day 9, these were seeded onto coverslips coated with fibrinogen and fibronectin and uncoated coverslips. Half were treated with 6.25 mM NIC. I had extra cells, so I also seeded the following conditions: 

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3 cell densities: 100,000 cells/mL, 75,000 cells/mL, 50,000 cells/mL Poly-L-lysine coated coverslips and fibronectin-coated coverslips 2 sets for Wright-Giemsa and beta-tubulin staining

All cells were observed over the next few days and Wright-Giemsa and β-tubulin fluorescence staining was done on Day 14. Protein Coating & Staining

Protocols for Wright-Giemsa and β-tubulin fluorescence staining can be found in Appendix A. Volumes were adjusted based on the size of the well or coverslip being used in each experiment. Protocols for ECM protein coating are based on recommendations from vendors and are included in Appendix B. Polydopamine Coating 96-well plates were coated with polydopamine. A 5 mg/mL solution of dopamine-HCl was prepared in 10 mM TRIZMA (pH 8.5), and 100 μL was added to each well. The well plate was sealed and placed in a sonicating water bath for 6 hours, then rinsed with MilliQ water. FibraCel discs (New Brunswick Scientific) were used in some trials; these were added to the 96-well plates and coated with pDA using the same protocol. PDA Functionalization with NeutrAvidin NeutrAvidin was added to pDA-coated wells at varying concentrations from 0.1 mg/mL to 0.02 mg/mL. 100 μL was added to each well and let sit overnight at room temperature. Supernatants were collected and the Coomassie Plus protein assay (Pierce/Thermo Scientific) was performed on the supernatants to determine how much protein had not bound the pDA. The amount of bound protein was deduced from the results of the assay. Results CHRF Cell Line experiment Protein coated Untreated

Untreated

Protein coated PMA-treated

PMA-treated

CHRF cells cultured in IMDM + 10% FBS  240,000 cells in 3.2 mL media seeded per well Polystyrene 6-well plates  Four plates coated with fibronectin, vitronectin, Type I collagen, or fibrinogen Staining and imaging done at Day 8 Images taken at 40X magnification

Images from CHRF Cell Line experiment:

Figure 3. CHRF cells, PMA-treated and controls, on fibronectin-coated wells and polystyrene controls.

Figure 5. CHRF cells, PMA-treated and controls, on Type I collagen-coated wells and polystyrene controls.

Figure 4. CHRF cells, PMA-treated and controls, on vitronectin-coated wells and polystyrene controls.

Figure 6. CHRF cells, PMA-treated and controls, on fibrinogen-coated wells and polystyrene controls.

Primary Murine Megakaryocyte Experiment

Fibrinogen Tpo only

Vitronectin Tpo only

Fibrinogen Tpo+NIC

Vitronectin Tpo+NIC

Fibronectin Tpo only

Fibronectin Tpo+NIC

Primary Mks were cultured in DMEM + 10% FBS + 1% pen/strep, supplemented with rhTpo, rhSCF, and rmIL-3  Half the wells were treated with 12.5 mM NIC  1 x 106 cells/mL in 3.2 mL media seeded per well Polystyrene 6-well plates coated with fibrinogen, fibronectin, and vitronectin Wright-Giemsa staining and imaging done at Day 4

Images from Primary Murine Megakaryocyte Experiment

Figure 7. Images of primary murine megakaryocytes cultured with Tpo and treated +/- NIC; fibronectin, fibrinogen, and vitronectin coated surfaces compared to polystyrene controls. Tpo+NIC condition on polystyrene has been enlarged to show scale; scale bar on bottom left corner represents 15 microns.

Results: Primary Human Megakaryocyte Experiment – Trial 1 Day 13 primary Mks cultured in media supplemented with Tpo, SCF, IL11, IL-6, and heparin Half of the wells were treated with 6.25 mM NIC  100,000 cells/mL, 3.2 mL seeded per well Polystyrene 6-well plates coated with fibrinogen, fibronectin, and vitronectin Wright-Giemsa staining and imaging done At Day 20 of this primary human megakaryocytic culture, a co-stain for CD41 and CD62p was performed and analyzed on the flow cytometer to look for platelets. No platelets were seen with this assay. It was done using 1 mL of suspended cells from each well. The remaining cultures continued and were observed over the next few days. Some proplatelet formation was seen later on, and the cells were stained at Day 28. This is a longer time period than is usual for these cells, and the cells in the NIC-treated wells were not healthy by this point. In the remaining wells, there were some long proplatelet extensions, especially in the fibrinogen- and fibronectin-coated wells. Before this culture, previous cultures of primary human megakaryocytes were not successful in producing proplatelets to this degree.

All treated with Tpo+cytokines, without NIC

Figure 8. Images of primary human Mks; stained at Day 28. Polystyrene and Fibronectin images are at 20x; Fibrinogen image is at 40x.

Results: Primary Human Megakaryocyte Experiment – Trial 2 Proplatelets began forming very quickly, and a few were visible already after one day. At Day 11, proplatelet formation was not much different than it had been at Day 8. Fibronectin and fibrinogen both had good adhesion, and fibrinogen had the most cells showing proplatelet extensions. On Day 12, which was 5 days after NIC treatment, the cells were stained with Wright-Giemsa. Although proplatelet formation was still very minimal, this culture showed more proplatelet formation than previous cultures of primary human Mks. Day 7 primary Mks cultured in media supplemented with Tpo; dead cell removal had been done at Day 2 Half of the wells were treated with 6.25 mM NIC  80,000 cells/mL, 2 mL seeded per well Polystyrene 6-well plates coated with fibrinogen and fibronectin Wright-Giemsa staining and imaging done at Day 12 Images taken at 20X

Polystyrene; Tpo+NIC

Fibronectin; Tpo+NIC

Fibrinogen; Tpo+NIC Figure 9. Images of primary human megakaryocytes on polystyrene, fibronectin, and fibrinogen surfaces, treated with Tpo and NIC. Cells were stained at Day 12, 5 days after NIC treatment.

Results: Primary Human Megakaryocyte Experiment – Trial 3 These cells did not show any proplatelets throughout the culture period. They were seeded onto protein surfaces at Day 12 and by Day 20, proplatelet formation was not apparent. WrightGiemsa staining was done at this point because cells had begun to die off, but no proplatelets were seen in the images either. DISCUSSION: One possible explanation is the low volume that was used in this experiment due to the total number of cells available. Additionally, they were seeded at Day 12 which is quite late in the culture, so NIC probably did not have much of an effect.

Results: Primary Human Megakaryocyte Experiment – Trial 4 Cells seeded on Day 5 The cells were observed at 2-hour intervals twice on the day they were seeded, and once or twice a day on following days. The cells seeded at Day 5 showed some short extensions by Day 8, but the majority of the cells were not showing much proplatelet formation. NIC-treated wells had a lot of larger cells by Day 9 of culture. Protein-coated wells showed good cell adhesion, and some wells had very high cell densities, which made it difficult to distinguish detail. At Day 11, the FN coated wells that were treated with NIC showed many more large cells compared to the FG and control wells. These cells were stained at Day 12, because they were beginning to die off at that point. Images were taken at 20x. Cells seeded on Day 8 These cells began to show large cells and good adhesion two days after seeding onto surfaces. At Day 10, many cells, especially in the Tpo only and good expansion wells, showed a lot of large cells in NIC treated wells and many were starting to show proplatelet extensions. At Day 12, there was a lot of good proplatelet formation in NIC treated wells on all surfaces, with the most being visible in the Tpo only and good expansion wells. These were also stained on Day 12. Images were taken at 20x.

Figure 00. Cells seeded at Day 8 on fibrinogen; cytokines: Tpo, SCF, IL-11, IL-3. NIC-treated. Proplatelet extensions can be seen within large cell clumps.

Figure 11. Cells seeded at Day 8 on fibronectin; cytokines: Tpo, SCF, IL-11, IL-3. NIC-treated. Some proplatelet extensions, both short and long, visible on several cells.

Figure 12. Cells seeded at Day 8 on uncoated coverslip; cytokines: Tpo, SCF, IL-11, IL-3. NIC-treated. No proplatelet formation is visible and not very many cells adhered to the surface.

Wright-Giemsa images were generally consistent with the pre-stain observations. The cells grown in Tpo, SCF, IL-11, and IL-3 (cytokines promoting good Mk expansion) showed considerably more proplatelet formation than the other two cytokine combinations. Fluorescence stained coverslips did not show proplatelet formation aside from a few cells with only one or two very short extensions. Fibronectin and fibrinogen showed many more cells displaying proplatelet extensions compared to the uncoated coverslips. Before staining, the uncoated coverslip for this condition showed 1 or 2 cells with some proplatelet extension, but those may have washed away during staining, as they could not be found afterward. Discussion:Also, the only cells I could see were all toward the outer edges of the coverslip. All I can think of is that the volumes of antibodies I used were not sufficient to reach all the cells, because pre-staining, there was proplatelet formation in some of the wells. I used 50 microliters of 2.5 microgram/mL of primary antibody and 50 microliters of a 1:100 dilution of secondary antibody to stain. Swapna and I decided on 25 microliter volumes for the chamber slides I had used in previous experiments based on Lisa’s protocol, and I doubled it based on the area of the coverslip compared to the area of the chamber wells. I can try increasing volumes when I stain in the next experiment.

Results: Primary Human Megakaryocyte Experiment – Trial 5

Figure 13. Cells seeded on fibrinogen at Day 9 & stained at Day 14; cytokines: Tpo, SCF, IL-3, IL-11. NIC-treated. Scale bar represents 30 microns.

Figure 14. Cells seeded on fibrinogen at Day 9 & stained at Day 14; cytokines: Tpo, SCF, IL-6, IL-11, IL-3. NIC-treated. Scale bar represents 30 microns.

Figure 15. Cells seeded on fibronectin at Day 9, staining done on Day 14; cytokines: Tpo, SCF, IL-11, IL-3. NIC-treated. Scale bar represents 30 microns.

Figure 16. Cells seeded on fibronectin at Day 9, staining done on Day 14; cytokines: Tpo, SCF, IL-11, IL-3, IL-6. NIC-treated. Scale bar represents 30 microns.

Figure 17. (a) Cells seeded at Day 9 on uncoated coverslip with Tpo, SCF, IL-11, IL-3. NIC-treated. (b) Same conditions but with cytokines Tpo, SCF, IL-11, IL-6, IL-3. Scale bars represent 30 microns.

Overall, the expansion cytokines (Tpo, SCF, IL-11, IL-3) in combination with either fibronectin or fibrinogen gave the best results, which is consistent with the previous experiment. I found the most cells displaying proplatelets for these conditions. There is some proplatelet formation in the 75,000- and 50,000-cell wells, on fibronectin. The poly-L-lysine seems to help with recovery after staining. These images can be found in Appendix C. Results: NeutrAvidin (NA) on Polydopamine-coated Surfaces

The Coomassie Plus assay has been tested with NA on pDA-coated 96-well plates, but the amount of NA that bound to a well was too little to be detected by the assay. To increase the available surface area, FibraCel discs were inserted into the well, and coated with pDA. Figure 18 shows wells containing pDA-coated FibraCel discs.

Figure 08. Polydopamine-coated FibraCel discs in a 96-well plate.

The Coomassie Plus assay (Micro protocol) was done on supernatants from the NA binding step to determine how much NA came out in solution, from that, amount of bound NA was calculated by multiplying the concentration given by the assay with the volume of supernatants plus rinses. When 5 μg of NA was added to a well, an average of 1.35 μg bound and when 2 μg of NA was added, an average of 1.65 μg bound. NA Micro Sta ndards

N e u trA vid in b o u n d to p DA -co a te d F ib ra Ce l d iscs

0.3 y = -0.0003x2 + 0.0178x + 0.0014 R2 = 0.9986

2

0.2 0.15 0.1 0.05 0 0

5

10

15

20

25

30

NA conce ntration (ug/m L)

Bound protein (ug)

Absorbance @ 570 nm

0.25

1.5 1 0.5 0 2 ug N A added overnight

5 ug N A added overnight

A ve ra g e a cro ss 16 a n d 22 w e lls

Figure 09. Standard curve of NeutrAvidin solutions for Coomassie Plus assay, micro protocol. Absorbances were read at 570 nm.

Figure 20. Amount of NA bound to pDA-coated FibraCel discs after 2 ug or 5 ug was added overnight.

Discussion CHRF Cell Line Experiment Primary Murine Megakaryocyte Experiment Primary Human Megakaryocyte Experiment Trial 1 Primary Human Megakaryocyte Experiment Trial 2 Primary Human Megakaryocyte Experiment Trial 3 Primary Human Megakaryocyte Experiment Trial 4 Primary Human Megakaryocyte Experiment Trial 5 NeutrAvidin on Polydopamine-coated Surfaces References 1. Miller, W. M. Bioengineering Challenges for Platelet Production from Hematopoietic Stem Cells. NIH Proposal (2008). 2.

Giammona, L.M., Fuhrken, P. G., Papoutsakis, E. T., & Miller, W. M. Nicotinamide (vitamin B3) increases the polyploidisation and proplatelet formation of cultured primary human megakaryocytes. British Journal of Haematology 135:4, 554-566 (2006).

3.

Larson, M. K. & Watson, S. P. Regulation of proplatelet formation and platelet release by integrin αIIbβ3. Blood 108, 1509-1514 (2006).

4.

Balduini, A., Pallotta, I., Malara, A., Lova, P., Pecci, A., Viarengo, G., Balduini, C. L., & Torti, M. Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes. Journal of Thrombosis and Haemostasis 6:11, 19001907 (2008).

5.

Garcia, A.S. Characterization of Ligand-Presenting Surfaces for Cell Culture Applications: The Effects of Lateral Mobility on Cell Adhesion and Surface Stability. PhD Dissertation, Northwestern University (2009).

6.

Lee, H., Dellatore, S. M., Miller, W. M., & Messersmith, P. B. Mussel-inspired Surface Chemistry for Multifunctional Coatings. Science 318:5849, 426-430 (2007).

7. Tables & Figures Supplemental Data Gantt Chart

Appendix A: Staining Protocols Wright-Giemsa staining (for a 24-well plate): 1. Gently remove media from wells. 2. Rinse gently with PBS. 3. Add 0.15 mL methanol and fix for 20 seconds. 4. Remove methanol. 5. Add 0.15 mL Wright-Giemsa stain for 45 seconds. 6. Remove stain. 7. Destain for 45 seconds in DI water at least 5 times. Gently swirl to remove excess stain. 8.

Add Cytoseal mounting media and adhere to glass slide; store at 4°C until imaging.

Beta-tubulin staining (for 24-well plate): 1. Add 0.667 mL 10% paraformaldehyde directly to media and fix for 15 minutes. 2.

Remove media and add 500 µ L 0.3% Triton X-100 in PBS to permeabilize for 5 minutes.

3.

Incubate for 1 hour with 500 µ L 2% BSA in PBS + 10% goat serum to block.

4.

Drain solution and incubate with 200 µ L primary antibody (BD Pharmingen, Purified Mouse Anti-beta-tubulin, 2.5 µ g/mL) in PBS + 2% BSA for 1 hour at room temperature.

5. Wash cells in PBS+0.1 M glycine 3 times. 6.

Incubate with 250 µ L secondary antibody (FITC conjugated Goat anti-mouse IgM, 1:100 dilution) in PBS + 2% BSA + 2% goat serum for 1 hour in the dark.

7. Wash cells in PBS+0.1 M glycine 3 times. 8.

Add DAPI/mounting media to coverslip on glass slide. Store at –20°C until imaging.

Appendix B: ECM Protein Coating Protocols 

Human Fibrinogen (Innovative Research) Prepare a 10 μg/mL solution of fibrinogen in PBS and add 1 mL per well in a 6-well plate, or 300 μL per well in a 24-well plate. Incubate at room temperature overnight. Rinse 3 times with PBS. Store unused plates at 4ºC for up to two weeks.

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Human Fibronectin (Sigma) Prepare a 30 μg/mL solution of fibronectin in Hank’s Balanced Salt Solution (pH 7.1). Add 300 μL per well in a 24-well plate. Incubate for at least 45 minutes at room temperature. Rinse with HBSS or PBS. Store unused plates at 4ºC for up to two weeks.

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Human Vitronectin (Sigma) Prepare a 1 μg/mL solution of vitronectin in sterile water. Add 1 mL per well in a 6-well plate. Incubate at 37ºC for 2 hours. Rinse with HBSS. Store unused plates at 4ºC for up to two weeks.

Appendix C: Additional Images from Primary Human Mk Experiment, Trial 5 These images are from coverslip coated with poly-L-lysine or fibronectin. Cell densities of 50,000 cells/mL and 75,000 cells/mL were tested on these coverslips. These cells came from the group treated with Tpo, SCF, IL-11, and IL-3, as well as nicotinamide.