Covalent immobilization of stem cell factor and stromal derived factor 1α for in vitro culture of hematopoietic progenitor cells

Abstract

Hematopoietic stem cells (HSCs) are currently utilized in the treatment of blood diseases, but widespread application of HSC therapeutics has been hindered by the limited availability of HSCs. With a better understanding of the HSC microenvironment and the ability to precisely recapitulate its components, we may be able to gain control of HSC behavior. In this work we developed a novel, biomimetic PEG hydrogel material as a substrate for this purpose and tested its potential with an anchorage-independent hematopoietic cell line, 32D clone 3 cells. We immobilized a fibronectin-derived adhesive peptide sequence, RGDS; a cytokine critical in HSC self-renewal, stem cell factor (SCF); and a chemokine important in HSC homing and lodging, stromal derived factor 1α (SDF1α), onto the surfaces of poly(ethylene glycol) (PEG) hydrogels. To evaluate the system's capabilities, we observed the effects of the biomolecules on 32D cell adhesion and morphology. We demonstrated that the incorporation of RGDS onto the surfaces promotes 32D cell adhesion in a dose-dependent fashion. We also observed an additive response in adhesion on surfaces with RGDS in combination with either SCF or SDF1α. In addition, the average cell area increased and circularity decreased on gel surfaces containing immobilized SCF or SDF1α, indicating enhanced cell spreading. By recapitulating aspects of the HSC microenvironment using a PEG hydrogel scaffold, we have shown the ability to control the adhesion and spreading of the 32D cells and demonstrated the potential of the system for the culture of primary hematopoietic cell populations.

Keywords: Hematopoietic cell; Hydrogel; Poly(ethylene glycol); Stem cell factor; Stromal derived factor 1α.

Figure 1. Microfabrication and Functionalization of PEG-DA hydrogel wells

A. To generate PEG-DA wells, SU-8 2100 photoresist was spin-coated onto glass slides. The photoresist slab was then exposed to UV light through a high resolution photomask. After removing the unexposed photoresist, PEG-DA wells were molded between a glass slides and the slide containing the SU-8 pillars. B. The bottom surfaces of the wells were biofunctionalized by pipetting an aqueous solution containing PEGylated biomolecules and a photoinitiator into the wells and exposing the material to UV light for 3 minutes. (Note: not to scale)

Figure 2. Western blot confirming PEGylation of SCF and SDF1α

A. In the SCF lane, a band at 18 kDa corresponds to the molecular weight (MW) of the extracellular domain of SCF. In the PEG-SCF lane, there is a smear beginning at a MW of ~28 kDa. The increase in MW confirms the addition of PEG chains to the SCF molecule, and the resulting smear indicates that some SCF molecules have multiple PEG chains. B. There is a band present in the SDF1α lane near 8 kDa. In the PEG-SDF1α lane you can see a smear beginning around 14 kDa indicating the protein has been successfully conjugated to PEG chains.

Figure 3. Bioactivity of PEGylated SCF and SDF1α

The bioactivity of the PEGylated proteins was evaluated by culturing 32D cells in media supplemented with either no protein, the natural occurring form of the protein, or the PEGylated version of the protein. For SCF, the effects on 32D cell proliferation and for SDF1α the effects on 32D cell migration were evaluated. A. In the SCF bioactivity assay, the percent change in 32D cell number after 5 days was significantly higher when SCF or PEG-SCF was added to the media. We saw no significant difference between the PEGylated and unPEGylated versions of the protein (n=8). B. In the migration assay, when SDF1α or PEG-SDF1α was added to the media, we saw an increase in 32D cell migration. However, the PEG chains appeared to interfere with the protein function as the migration of 32D cells in the presence of PEG-SDF1α was significantly reduced compared to that demonstrated with SDF1α (n=54). Data reported as average ± standard deviation, * denotes significance compared to control, and # denotes significance compared to PEG-SDF1α (p<0.05).

Figure 4. Adherent 32D cells on hydrogel well surfaces functionalized with biomolecules

32D cells were cultured on surfaces with covalently immobilized RGDS, SCF, and SDF1α. As the concentration of RGDS was increased, there was a corresponding significant increase in cell adhesion. With the addition of SCF (400 ng/cm²) or SDF1α (400 ng/cm²) to the surface of gels with medium RGDS concentrations (25 μg/cm²), there was a significant increase in cell adhesion compared to the peptide alone. However, with 250 μg/cm² RGDS on the surface, the addition of SCF or SDF1α did not similarly increase the number of adherent cells. Data presented as average ± standard deviation, * indicates statistical significance compared to 0 μg/cm² RGDS, 2.5 μg/cm² RGDS, and 25 μg/cm² RGDS (n=4–8, p<0.05).

Figure 5. 32D cell morphology in bioactive PEG hydrogel wells

Representative images of 32D cells on hydrogel surfaces. Actin filaments are stained with phalloidin (green) and nuclei are stained with DAPI (blue). Higher concentrations of RGDS on the surface resulted in more adherent 32D cells on the gels. With the covalent incorporation of SCF and SDF1α on the surfaces, the cells appeared more spread, and distinct filopodia can be seen extending from many of the cells (denoted by white arrows). (A. PEG-DA, B. 2.5 μg/cm² RGDS, C. 25 μg/cm² RGDS, D. 250 μg/cm² RGDS, E. 25 μg/cm² RGDS + 400 ng/cm² SCF, F. 25 μg/cm² RGDS + 400 ng/cm² SDF1α; Scale bars = 25 μm).

Figure 6. 32D cell morphology in TCPS wells with physioadsorbed proteins

Representative images of 32D cells on hydrogel surfaces. Actin filaments are stained with phalloidin (green) and nuclei are stained with DAPI (blue). Cells appeared rounded and small in all groups. (A. 1 μg/cm² FN, B. 1 μg/cm² FN + 400 ng/cm² SCF, C. 1 μg/cm² FN + 400 ng/cm² SDF1α, D. 400 ng/cm² SCF, E. 400 ng/cm² SDF1α; Scale bars = 25 μm).

Figure 7. Quantification of 32D cell morphology in hydrogel and TCPS wells

A) 32D cells spread to a greater extent on hydrogels modified with RGDS (25 μg/cm²) and SCF (400 ng/cm²) or SDF1α (400 ng/cm²) compared to RGDS alone gels and TCPS wells with physioadsorbed proteins. The wide distribution of cell size is due to the heterogeneity of 32D cells. (Black bars are hydrogel wells while grey bars are TCPS wells. Bars represent mean ± standard deviation. Bars that do no share a common letter are significantly different, n=105 (0 μg/cm² RGDS), 193 (2.5 μg/cm² RGDS), 2454 (25 μg/cm² RGDS), 863 (250 μg/cm² RGDS), 617 (SCF), 631 (SDF1α), 622 (1 μg/cm² FN), 1177(1 μg/cm² FN +SCF), 839(1 μg/cm² FN +SDF1α), 881(SCF), 472(SDF1α), p < 0.05).

Figure 8. 32D cell area distribution

A. Cell area distribution on hydrogel wells. On all three RGDS concentrations most cells were between 100 – 200 μm². On hydrogel surfaces with SCF or SDF1α there was a shift to the right and a broadening of the cell area distribution compared to RGDS alone. B. Cell area distribution on TCPS wells. On physioadsorbed biomolecules, cell area was centered around 100 – 200 μm². The distribution of cell sizes is not as wide as on hydrogel surfaces with most cells smaller than 400 μm².

Figure 9. Circularity of 32D cells in PEG hydrogel and TCPS wells

32D cells on samples with no RGDS present retained a round shape. 32D cells on bioactive hydrogels were less circular indicating spreading. 32D cells on TCPS coated plates were rounder and less spread than cells on hydrogels with covalently immobilized SCF or SDF1α. Black bars are hydrogel wells while grey bars are TCPS wells. Bars that do no share a common letter are significantly different. n=105 (0 μg/cm² RGDS), 185 (2.5 μg/cm² RGDS), 2454 (25 μg/cm² RGDS), 699 (250 μg/cm² RGDS), 497 (25 μg/cm² RGDS+SCF), 458 (25 μg/cm² RGDS+SDF1α), 482 (1 μg/cm² FN), 861 (1 μg/cm² FN +SCF), 617 (1 μg/cm² FN +SDF1α), 625 (SCF), 388 (SDF1α) (p < 0.05).