Hyaluronan based hydrogels provide an improved model to study megakaryocyte-matrix interactions

Abstract

Hyaluronan (HA) is a glycosamminoglican involved in cell biology as well as a relevant polymer for tissue engineering and regenerative medicine. Megakaryocytes (Mks) are immersed in a mesh of extracellular matrix (ECM) components that regulate their maturation in the bone marrow (BM) and the release of platelets into the bloodstream. While fibrous ECMs such as collagens and fibronectin have been demonstrated to differently regulate Mk function and platelet release, the role of HA, that fills the majority of the BM extracellular interstitial space, has not been investigated so far. Here we demonstrated that, although human Mks express HA receptors, they are not affected by HA in terms of in vitro differentiation, maturation and platelet formation. Importantly, chemical properties of HA were exploited to generate hydrogels with entrapped ECMs that represent a useful model to more closely mimic the tridimensional characteristics of the BM environment for studying Mk function. In conclusion, in this work we demonstrated that HA is an ideal candidate for a 3D ex vivo model of human BM ECM component environment.

Keywords: Bone marrow; Extracellular matrix; Hyaluronic acid; Hydrogels; Megakaryocytes; Proplatelet formation; Proteoglycans; Thrombopoiesis.

Figure 1. Human Megakaryocytes express hyaluronan (HA) and HAS isoenzymes necessary for its biosynthesis

A) Real Time qPCR analysis of HAS1, HAS2 and HAS3 expression in cord blood derived CD34+ HSCs and differentiated Mks. Results are expressed as fold increase of HAS isoenzymes expression in Mks with respect to CD34+ HSCs. β2 microglobulin gene expression was used for the comparative concentration analysis. At least three independent experiments were performed. n.d. = not detectable. B) Western blotting analysis of HAS3 expression in human fibroblasts and differentiated Mks. C) Western blotting analysis of HAS3 expression in differentiated Mks and human peripheral blood platelets. D) Densitometry analysis of HAS3 levels in Mks and peripheral blood platelets. β-actin was used as internal control of protein loading. At least three independent experiments were performed. E) Immunofluorescence staining of HAS-3 (red) (middle panel) in TPO differentiated CD61+ (green) (left panel) Mks. 1×10⁵ Mks at day 13 of culture were cytospun onto poly-L-lysine coated glass coverslips and processed for immunofluorescence. Composite of images is shown on the right panel. Scale Bar=20μm. F) Immunofluorescence staining of hyaluronan (red) (middle panel) in TPO differentiated CD61+ (green) (left panel) Mks. Composite of images are shown on the right panel. 1×10⁵ Mks at day 13 of culture were cytospun, fixed and permeabilized or not with Triton X-100 to demonstrate the intracellular localization of HA. Cells were stained with Hyaluronan Binding Protein (HABP) and CD61 antibody, followed by Alexa Fluor 594 coniugated streptavidin and Alexa Fluor 488 conjugated antibody, respectively. Treatment with hyaluronidase was performed before Mks staining to confirm the presence of intracellular HA. Scale Bar=10μm. Hoechst 33258 was used to highlight nuclei (blue).

Figure 2. Hyaluronan receptors are differently expressed during hematopoietic stem cell differentiation into mature Mks

A) Western blotting analysis of CD44 and RHAMM expression in Mk lysates at day 7, 10 and 13 of differentiation. Actin was revealed to correct minor changes in protein loading. B) Immunofluorescence staining of RHAMM (red) in permeabilized (upper panels) and non permeabilized CD61 (green) positive Mks (lower panels). Scale Bar=30μm. C) FACS analysis of RHAMM staining in permeabilized (left panel) and non permeabilized (right panel) CD61 positive Mks. 2×10⁵ cells were harvested from cultures and analyzed by FACS. D) Representative FACS analysis of CD44 and RHAMM in mature CD61+ Mks. Percentages of CD44/CD61 or RHAMM/CD61 double positive cells are shown. E) Quantification of CD61/CD44 and CD61/RHAMM double positive cells percentages at day 13 of differentiation by FACS. At least three independent experiments were performed. Scale Bar=30μm.

Figure 3. Hyaluronan does not influence Mk differentiation, maturation and function

A) Mks were differentiated for 13 days in the presence of TPO (10 ng/mL) and three different concentration of high molecular weight HA (average 5000 kDa), in particular 10, 50 and 100 μg/mL. Mk output is defined as the normalized percentage of CD61 positive cells at the end of cultures with respect to TPO treated cells. At least three independent experiments were performed. B) Quantification of HA effects on Mk ploidy. 2×10⁵ untreated (Ctrl) or HA treated Mks were collected at day 13 of differentiation and analyzed by FACS after propidium iodide staining. At least three independent experiments were performed. C) Quantification of in vitro Mk-forming proplatelets in TPO and HA stimulated Mks. 1×10⁵ Mks were collected at day 13 of differentiation and seeded onto fibrinogen coated glass coverslips in the presence of TPO or TPO plus HA at different concentrations. After 16 hours, adherent cells were fixed, permeabilized and stained with α-tubulin antibody. Results are expressed as the normalized percentage of Mk-displaying proplatelets with respect to TPO treated cultures. D) Representative western blotting analysis of AKT and ERK 1/2 phosphorylation levels in TPO and TPO plus HA (100μg/ml) treated Mks. CD34+ HSC were purified from cord blood and cultured for 13 days in the presence of TPO (Ctrl) or TPO plus 100μg/ml of HA. Cells at day 13 were collected and lysed. Total AKT, ERK 1/2 were revealed to ensure equal protein loading. E) Densitometry analysis of pAKT/AKT and pERK/ERK ratios in TPO or TPO plus HA treated cultures of at least three independent experiments.

Figure 4. Hyaluronan hydrogel-based scaffolds represent an ideal model for studying 3D megakaryocyte interactions with other fibrous extracellular matrix proteins

A) Front (upper panel) and side (bottom panel) photomicrographs of hyaluronan hydrogels. HA was methacrylated and photocrosslinked using Irgacure 2959. Scale bar=7mm and 2mm, respectively. B) Mks were entrapped into methacrylated HA (MeHA) and hydrogels were seeded in culture media with TPO for 16 hours. Cell viability was evaluated by confocal microscopy after cell staining with a live (green)/dead (red) cell staining kit. Scale Bar=50μm. C) Cross section analysis of Z-stack of Mks encapsulated into methacrylated HA hydrogels as measured by confocal microscopy. Scale bar=200μm. D) Cartoon showing strategy adopted for the generation of MeHA hydrogels containing ECM components, such as type I and IV collagens and fibronectin (5 μg each). E) Phase contrast evaluation of proplatelet formation by human Mks in MeHA and MeHA plus ECM components hydrogels after 16 hours of culture. Scale Bar=20μm. F) Quantification of percentages of Mk-displaying proplatelets with respect to total Mks in MeHA hydrogels containing ECM components. At least 100 Mks per conditions were counted and three independent experiments were performed. p value<0.001.