Bioactive hydrogel scaffolds for controllable vascular differentiation of human embryonic stem cells

Abstract

We propose a new methodology to enhance the vascular differentiation of human embryonic stem cells (hESCs) by encapsulation in a bioactive hydrogel. hESCs were encapsulated in a dextran-based hydrogel with or without immobilized regulatory factors: a tethered RGD peptide and microencapsulated VEGF(165). The fraction of cells expressing vascular endothelial growth factor (VEGF) receptor KDR/Flk-1, a vascular marker, increased up to 20-fold, as compared to spontaneously differentiated embryoid bodies (EBs). The percentage of encapsulated cells in hydrogels with regulatory factors expressing ectodermal markers including nestin or endodermal markers including alpha-fetoprotein decreased 2- or 3-fold, respectively, as compared to EBs. When the cells were removed from these networks and cultured in media conditions conducive for further vascular differentiation, the number of vascular cells was higher than the number obtained through EBs, using the same media conditions. Functionalized dextran-based hydrogels could thus enable derivation of vascular cells in large quantities, particularly endothelial cells, for potential application in tissue engineering and regenerative medicine.

Figure 1. Hydrogel properties and cell attachment in the surface of dextran-based hydrogels

A) Schematic representation for the preparation of dextran-based hydrogels. B) Swelling (white columns) and elastic modulus (black columns) of hydrogels with different compositions (Average ± S.D., n=3). 20 μm microparticles were used in these assays. C) Representative light micrographs of attached hESCs after 24 h for dextran-based hydrogels with no Acr-PEG-RGD (C.1), 0.5 mM Acr-PEG-RGD (C.2), and 5 mM Acr-PEG-RGD (C.3). Bar corresponds to 100 μm.

Figure 2. Distribution and viability of hESC aggregates encapsulated within bioactive dextran-based hydrogels

A) Scanning electron micrographs of 7 μm (A.1, A.2) and 20 μm (A.3, A.4) PLGA microparticles at day 0 (A.1, A.3) and 10 (A.2, A.4), during the release of VEGF₁₆₅. B) Release profile of VEGF₁₆₅ from 7 μm (□) and 20 μm (○) microparticle formulations. C) Distribution of hESC aggregates on dextran-based hydrogels with 0.5 mM Acr-PEG-RGD and 5 mg/mL VEGF-loaded 20 μm microparticles, at day 0 (C.1, C.2) and day 10 (C.3,C.4). Top (C.1, C.3) and side (C.2, C.4) views. Arrows in C.3 and C.4 indicate clump of cells proliferating outside the hydrogel. Inset in C.3 shows the incorporation of microparticles (arrow) within the hESC aggregates. Scale bar corresponds to 200 μm. D) Mitochondrial metabolic activity (average ± SD, n=6) of hESC aggregates encapsulated in dextran-based hydrogels with no Acr-PEG-RGD (1), 0.5 mM Acr-PEG-RGD (2), 5 mM Acr-PEG-RGD (3), 0.5 mM Acr-PEG-RGD with 5 mg/mL of blank microparticles (4), and 0.5 mM Acr-PEG-RGD with 5 mg/mL of VEGF₁₆₅ loaded 20 μm (5) or 7 μm (6) microparticles. The absorbance at 540 nm was measured after 1 day (black bars) and 10 days (white bars). Both absorbances were normalized by day 1 absorbance.

Figure 3. Expression of endothelial and undifferentiating stem cell markers on hESC aggregates encapsulated in dextran-based hydrogels and on EBs

A,B) Summary of FACS (A), RT-PCR (B.1) and qPCR (B.2) analysis of undifferentiating cells (1), EBs at day 10 (2), hESC aggregates encapsulated into dextran-based hydrogels without (3) or containing 0.5 mM (4) or 5 mM Acr-PEG-RGD (5), hESC aggregates encapsulated into dextran-based hydrogels containing 0.5 mM Acr-PEG-RGD and 5 mg/mL of microparticles loaded with [20 μm (7) and 7 μm (8)] or without [20 μm (6)] VEGF₁₆₅. Please note that samples 3 and 4 are reversed in all gels. C) FACS analysis of EBs grown in suspension for 10 days in differentiation medium containing 50 ngmL⁻¹ VEGF₁₆₅. For all FACS and qPCR analysis, values indicate average ± S.D., from at least 3 independent experiments. *, ** and *** denote statistical significance (P< 0.05, P< 0.01 and P< 0.001, respectively). In the graph for the CD34 marker, statistical analyses were performed between all conditions and undifferentiating hESCs.

Figure 4. Localization and organization of endothelial markers on EBs and on hESC aggregates encapsulated in dextran-based hydrogels

Confocal images of CD34⁺ (A,B), PECAM1⁺ (C,D), VE-CAD⁺ (E,F) and KDR/Flk-1⁺ (G,H) cells from EBs (A,C,E,G) and from hESC aggregates encapsulated in dextran-based hydrogels with 0.5 mM Acr-PEG-RGD [B (×10); D (×10)], with 5.0 mM Acr-PEG-RGD [F (×25)] and with 0.5 mM Acr-PEG-RGD containing 5 mgmL⁻¹ of 7 μm microparticles loaded with VEGF₁₆₅ [H (×10)].

Figure 5. Ectodermal and endodermal differentiation of hESC aggregates encapsulated in dextran-based hydrogels

A, B) Expression of α-fetoprotein (1,2,3) and nestin (4,5,6) as revealed by imunohistochemistry, in EBs (1,4) and hESC aggregates encapsulated in dextran-based hydrogels (2,5) or in networks containing 0.5 mM Acr-PEG-RGD and 5 mgmL⁻¹ 7 μm microparticles (3,6). Bar corresponds to 100 μm. For the quantitative analysis of antibody staining (B), the results are average ± S.D. from two different samples and the counts were obtained in at least two different sections per sample. C) RT-PCR analysis of endoderm (albumin and α-fetoprotein) and ectoderm markers (nestin, neurofilament 68 Kd) in undifferentiated cells (1), EBs at day 10 (2), hESC aggregates encapsulated in dextran-based hydrogels with no Acr-PEG-RGD (3), 0.5 mM Acr-PEG-RGD (4), 5 mM Acr-PEG-RGD (5), 0.5 mM Acr-PEG-RGD with 5 mg/mL of 20 μm blank microparticles (6), and 0.5 mM Acr-PEG-RGD with 5 mg/mL of 20 (7) or 7 μm (8) microparticles loaded with VEGF₁₆₅.

Figure 6. Expression of endothelial and undifferentiating stem cell markers in cells removed from hydrogel networks and further cultured in EGM-2 medium supplemented with VEGF₁₆₅

A) Scheme showing the protocols adopted to differentiate hESCs into the vascular lineage. (1) Undifferentiated hESCs cultured in EGM-2 medium supplemented with 50 ngmL⁻¹ VEGF₁₆₅ for 16 days. (2) Undifferentiated hESCs were induced to form EBs (see Materials and Methods), and the EBs grown in differentiation medium for 10 days. Then, single cells isolated from EBs were cultured in EGM-2 medium supplemented with 50 ngmL⁻¹ VEGF₁₆₅ for 6 additional days. (3). Undifferentiated hESCs were encapsulated in dextran-based hydrogels containing 0.5 mM Acr-PEG-RGD and 5 mgmL⁻¹ of microparticles with [20 μm (4) and 7 μm (5)] or without VEGF₁₆₅ [20 μm (3)], and cultured in differentiation medium for 10 days. Subsequently, single cells isolated from the encapsulated aggregates were cultured in EGM-2 medium supplemented with 50 ngmL⁻¹ VEGF₁₆₅ for 6 additional days. B) Summary of FACS analysis for endothelial and undifferentiating stem cells markers. Cells were differentiated according to protocol 1 (dashed columns), protocol 2 (grey columns) and protocol 3 (white (3), black (4) and dashed (5) columns). Values indicate average ± S.D. from two or three independent experiments (3 replicates per run). *. ** and *** denote statistical significance (P< 0.05, P< 0.01 and P< 0.001, respectively).