Promotion of hMDSC differentiation by combined action of scaffold material and TGF-ß superfamily growth factors.

Objective: Herein we propose a combined action of collagen type I (CA) or synthetic collagen-like-peptide functionalized with the cell adhesive RGD motif (PEG-CLP-RGD) hydrogels and selected growth factors to promote chondrogenic differentiation of human muscle-derived stem cells (hMDSCs) under normal and reduced oxygen conditions. Methods: hMDSCs were set for differentiation towards chondrogenic lineage using BMP-7 and TGF-ß3. Cells were seeded onto hydrogels loaded with growth factors (75ng/scaffold) and cultured for 28 days under normal (21%) and severe hypoxic (1%) conditions. Chondrogenesis was evaluated by monitoring collagen type II and GAG deposition, and quantification of ACAN expression by RT-PCR. Results: Sustained release of TGFß3 from the hydrogels was observed, 8.7 ± 0.5% of the initially loaded amount diffused out after 24 h from both substrates. For the BMP-7 growth factor, 14.8 ± 0.3% and 18.2 ± 0.6% of the initially loaded amount diffused out after 24 h from CA and CLP-RGD, respectively. The key findings of this study are: i) the self-supporting hydrogels themselves can stimulate hMDSC chondrogenesis by inducing gene expression of cartilage-specific proteoglycan aggrecan and ECM production; ii) the effect of dual BMP-7 and TGF-ß3 loading was more pronounced on CA hydrogel under normal oxygen conditions; iii) dual loading on PEG-CLP-RGD hydrogels did not have the synergistic effect, TGF-ß3 was more effective under both oxygen conditions; iv) BMP-7 can improve chondrogenic effect of TGF-ß3 on CA scaffolds, and hydrogels loaded with both growth factors can induce cartilage formation in hMDSC cultures. Conclusion: Our results support the potential strategy of combining implantable hydrogels functionalized with differentiation factors toward improving cartilaginous repair.

Comparison of Microglial Morphology and Function in Primary Cerebellar Cell Cultures on Collagen and Collagen-Mimetic Hydrogels.

Neuronal-glial cell cultures are usually grown attached to or encapsulated in an adhesive environment as evenly distributed networks lacking tissue-like cell density, organization and morphology. In such cultures, microglia have activated amoeboid morphology and do not display extended and intensively branched processes characteristic of the ramified tissue microglia. We have recently described self-assembling functional cerebellar organoids promoted by hydrogels containing collagen-like peptides (CLPs) conjugated to a polyethylene glycol (PEG) core. Spontaneous neuronal activity was accompanied by changes in the microglial morphology and behavior, suggesting the cells might play an essential role in forming the functional neuronal networks in response to the peptide signalling. The present study examines microglial cell morphology and function in cerebellar cell organoid cultures on CLP-PEG hydrogels and compares them to the cultures on crosslinked collagen hydrogels of similar elastomechanical properties. Material characterization suggested more expressed fibril orientation and denser packaging in crosslinked collagen than CLP-PEG. However, CLP-PEG promoted a significantly higher microglial motility (determined by time-lapse imaging) accompanied by highly diverse morphology including the ramified (brightfield and confocal microscopy), more active Ca2+ signalling (intracellular Ca2+ fluorescence recordings), and moderate inflammatory cytokine level (ELISA). On the contrary, on the collagen hydrogels, microglial cells were significantly less active and mostly round-shaped. In addition, the latter hydrogels did not support the neuron synaptic activity. Our findings indicate that the synthetic CLP-PEG hydrogels ensure more tissue-like microglial morphology, motility, and function than the crosslinked collagen substrates.

Cardiomyogenic Differentiation Potential of Human Dilated Myocardium-Derived Mesenchymal Stem/Stromal Cells: The Impact of HDAC Inhibitor SAHA and Biomimetic Matrices.

Dilated cardiomyopathy (DCM) is the most common type of nonischemic cardiomyopathy characterized by left ventricular or biventricular dilation and impaired contraction leading to heart failure and even patients' death. Therefore, it is important to search for new cardiac tissue regenerating tools. Human mesenchymal stem/stromal cells (hmMSCs) were isolated from post-surgery healthy and DCM myocardial biopsies and their differentiation to the cardiomyogenic direction has been investigated in vitro. Dilated hmMSCs were slightly bigger in size, grew slower, but had almost the same levels of MSC-typical surface markers as healthy hmMSCs. Histone deacetylase (HDAC) activity in dilated hmMSCs was 1.5-fold higher than in healthy ones, which was suppressed by class I and II HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) showing activation of cardiomyogenic differentiation-related genes alpha-cardiac actin (ACTC1) and cardiac troponin T (TNNT2). Both types of hmMSCs cultivated on collagen I hydrogels with hyaluronic acid (HA) or 2-methacryloyloxyethyl phosphorylcholine (MPC) and exposed to SAHA significantly downregulated focal adhesion kinase (PTK2) and activated ACTC1 and TNNT2. Longitudinal cultivation of dilated hmMSC also upregulated alpha-cardiac actin. Thus, HDAC inhibitor SAHA, in combination with collagen I-based hydrogels, can tilt the dilated myocardium hmMSC toward cardiomyogenic direction in vitro with further possible therapeutic application in vivo.

Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels.

Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell-cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca2+ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies.