A Combined Synthetic-Fibrin Scaffold Supports Growth and Cardiomyogenic Commitment of Human Placental Derived Stem Cells

Aims: A potential therapy for myocardial infarction is to deliver isolated stem cells to the infarcted site. A key issue with this therapy is to have at one\u27s disposal a suitable cell delivery system which, besides being able to support cell proliferation and differentiation, may also provide handling and elastic properties which do not affect cardiac contractile function. In this study an elastic scaffold, obtained combining a poly(ether)urethane-polydimethylsiloxane (PEtU-PDMS) semi-interpenetrating polymeric network (s-IPN) with fibrin, was used as a substrate for in vitro studies of human amniotic mesenchymal stromal cells (hAMSC) growth and differentiation. Methodology/Principal Findings: After hAMSC seeding on the fibrin side of the scaffold, cell metabolic activity and proliferation were evaluated by WST-1 and bromodeoxyuridine assays. Morphological changes and mRNAs expression for cardiac differentiation markers in the hAMSCs were examined using immunofluorescence and RT-PCR analysis. The beginning of cardiomyogenic commitment of hAMSCs grown on the scaffold was induced, for the first time in this cell population, by a nitric oxide (NO) treatment. Following NO treatment hAMSCs show morphological changes, an increase of the messenger cardiac differentiation markers [troponin I (TnI) and NK2 transcription factor related locus 5 (Nkx2.5)] and a modulation of the endothelial markers [vascular endothelial growth factor (VEGF) and kinase insert domain receptor (KDR)]. Conclusions/Significance: The results of this study suggest that the s-IPN PEtU-PDMS/fibrin combined scaffold allows a better proliferation and metabolic activity of hAMSCs cultured up to 14 days, compared to the ones grown on plastic dishes. In addition, the combined scaffold sustains the beginning of hAMSCs differentiation process towards a cardiomyogenic lineage

Numerical methods for the design and description of in vitro expansion processes of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction or inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hMSC-based therapies, in vitro expansion is necessary prior to the clinical application in order to achieve the required cell numbers. Safe, reproducible, and economic in vitro expansion of hMSCs for autologous and allogeneic therapies can be problematic because the cell material is restricted and the cells are sensitive to environmental changes. It is beneficial to collect detailed information on the hydrodynamic conditions and cell growth behavior in a bioreactor system, in order to develop a so called “Digital Twin” of the cultivation system and expansion process. Numerical methods, such as Computational Fluid Dynamics (CFD) which has become widely used in the biotech industry for studying local characteristics within bioreactors or kinetic growth modelling, provide possible solutions for such tasks. In this review, we will present the current state-of-the-art for the in vitro expansion of hMSCs. Different numerical tools, including numerical fluid flow simulations and cell growth modelling approaches for hMSCs, will be presented. In addition, a case study demonstrating the applicability of CFD and kinetic growth modelling for the development of an microcarrier-based hMSC process will be shown

A mathematical model of mechanotransduction reveals how mechanical memory regulates mesenchymal stem cell fate decisions

Abstract Background Mechanical and biophysical properties of the cellular microenvironment regulate cell fate decisions. Mesenchymal stem cell (MSC) fate is influenced by past mechanical dosing (memory), but the mechanisms underlying this process have not yet been well defined. We have yet to understand how memory affects specific cell fate decisions, such as the differentiation of MSCs into neurons, adipocytes, myocytes, and osteoblasts. Results We study a minimal gene regulatory network permissive of multi-lineage MSC differentiation into four cell fates. We present a continuous model that is able to describe the cell fate transitions that occur during differentiation, and analyze its dynamics with tools from multistability, bifurcation, and cell fate landscape analysis, and via stochastic simulation. Whereas experimentally, memory has only been observed during osteogenic differentiation, this model predicts that memory regions can exist for each of the four MSC-derived cell lineages. We can predict the substrate stiffness ranges over which memory drives differentiation; these are directly testable in an experimental setting. Furthermore, we quantitatively predict how substrate stiffness and culture duration co-regulate the fate of a stem cell, and we find that the feedbacks from the differentiating MSC onto its substrate are critical to preserve mechanical memory. Strikingly, we show that re-seeding MSCs onto a sufficiently soft substrate increases the number of cell fates accessible. Conclusions Control of MSC differentiation is crucial for the success of much-lauded regenerative therapies based on MSCs. We have predicted new memory regions that will directly impact this control, and have quantified the size of the memory region for osteoblasts, as well as the co-regulatory effects on cell fates of substrate stiffness and culture duration. Taken together, these results can be used to develop novel strategies to better control the fates of MSCs in vitro and following transplantation

A biomaterials approach to influence stem cell fate in injectable cell-based therapies

Background Numerous stem cell therapies use injection-based administration to deliver high-density cell preparations. However, cell retention rates as low as 1% have been observed within days of transplantation. This study investigated the effects of varying administration and formulation parameters of injection-based administration on cell dose recovery and differentiation fate choice of human mesenchymal stem cells. Methods The impact of ejection rate via clinically relevant Hamilton micro-syringes and biomaterial-assisted delivery was investigated. Cell viability, the percentage of cell dose delivered as viable cells, proliferation capacity as well as differentiation behaviour in bipotential media were assessed. Characterisation of the biomaterial-based cell carriers was also carried out. Results A significant improvement of in-vitro dose recovery in cells co-ejected with natural biomaterials was observed, with ejections within 2% (w/v) gelatin resulting in 87.5 ± 14% of the cell dose being delivered as viable cells, compared to 32.2 ± 19% of the dose ejected in the commonly used saline vehicle at 10 μl/min. Improvement in cell recovery was not associated with the rheological properties of biomaterials utilised, as suggested by previous studies. The extent of osteogenic differentiation was shown to be substantially altered by choice of ejection rate and cell carrier, despite limited contact time with cells during ejection. Collagen type I and bone-derived extracellular matrix cell carriers yielded significant increases in mineralised matrix deposited at day 21 relative to PBS. Conclusions An enhanced understanding of how administration protocols and biomaterials influence cell recovery, differentiation capacity and choice of fate will facilitate the development of improved administration and formulation approaches to achieve higher efficacy in stem cell transplantation

Multiscale fluid–structure interaction modelling to determine the mechanical stimulation of bone cells in a tissue engineered scaffold

Recent studies have shown that mechanical stimulation, by means of flow perfusion and mechanical compression (or stretching), enhances osteogenic differentiation of mesenchymal stem cells and bone cells within biomaterial scaffolds in vitro. However, the precise mechanisms by which such stimulation enhances bone regeneration is not yet clear. The physical environment within a scaffold under perfusion is extremely complex and requires a multiscale and multiphysics approach to study the mechanical stimulation of cells. In this study, we aim to determine the mechanical stimulation of osteoblasts seeded in a biomaterial scaffold under flow perfusion and mechanical compression using multiscale modelling by twoway fluid–structure interaction and FE approaches. The mechanical stimulation, in terms of wall shear stress (WSS) and strain in osteoblasts, is quantified at different locations within the scaffold for cells of different morphologies (i.e. attached, bridged). The results show that 75.4% of scaffold surface has a WSS of 0.1–10 mPa, which indicates the likelihood of bone cell differentiation at these locations. For attached and bridged osteoblasts, the maximum strains are 397 and 177,200με, respectively. Additionally, the results from mechanical compression show that attached cells are more stimulated (maximum strain = 22, 600 με) than bridged cells (maximum strain = 10, 000 με). Such information is important for understanding the biological response of osteoblasts under in vitro stimulation. Finally, a combination of perfusion and compression of a tissue engineering scaffold is suggested for osteogenic differentiation