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 ECM-
protein 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, Wright-
Giemsa 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.

 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.

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

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

 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

 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.

 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:

 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

CHRF cells cultured in IMDM + 10% FBS

Protein Protein  240,000 cells in 3.2 mL media seeded
coated
Untreated coated
PMA-treated per well
Polystyrene 6-well plates
 Four plates coated with fibronectin,
vitronectin, Type I collagen, or
fibrinogen
Untreated PMA-treated
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 Figure 4. CHRF cells, PMA-treated and controls, on
fibronectin-coated wells and polystyrene controls. vitronectin-coated wells and polystyrene controls.

Figure 5. CHRF cells, PMA-treated and controls, on Type I Figure 6. CHRF cells, PMA-treated and controls, on
collagen-coated wells and polystyrene controls. fibrinogen-coated wells and polystyrene controls.
Primary Murine Megakaryocyte Experiment

Primary Mks were cultured in DMEM +

10% FBS + 1% pen/strep,
Fibrinogen
supplemented with rhTpo, rhSCF,
Vitronectin Fibronectin
Tpo only Tpo only Tpo only 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 Vitronectin Fibronectin fibrinogen, fibronectin, and
Tpo+NIC Tpo+NIC Tpo+NIC 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, IL-
11, 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 All treated with
Tpo+cytokines,
At Day 20 of this primary human without NIC
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
Figure 8. Images of primary human Mks; stained at Day
wells were not healthy by this point. In the 28. Polystyrene and Fibronectin images are at 20x;
remaining wells, there were some long Fibrinogen image is at 40x.
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.
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 Polystyrene; Tpo+NIC Fibronectin; Tpo+NIC
per well
Polystyrene 6-well plates coated
with fibrinogen and
fibronectin
Wright-Giemsa staining and
imaging done at Day
12
Images taken at 20X

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. Wright-
Giemsa 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, Figure 17. (a) Cells seeded at Day 9 on uncoated
staining done on Day 14; cytokines: Tpo, SCF, coverslip with Tpo, SCF, IL-11, IL-3. NIC-treated.
IL-11, IL-3, IL-6. NIC-treated. Scale bar (b) Same conditions but with cytokines Tpo, SCF,
represents 30 microns. 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
0.25
R2 = 0.9986
2
Absorbance @ 570 nm

0.2

0.15 1.5
Bound protein (ug)

0.1

0.05 1
0
0 5 10 15 20 25 30
NA conce ntration (ug/m L) 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 Figure 20. Amount of NA bound to pDA-coated FibraCel discs after 2
protocol. Absorbances were read at 570 nm. 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, 1900-
1907 (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.

 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.

 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.