Isolation and characterisation of a porcine cardiac adherent cell population from heart biopsies

Cardiac diseases represent the major cause of mortality in the Western world. The discovery of the promising human Cardiac Adherent Proliferating (CAP) cells for a potential cardiac cell therapy by the Tissue Engineering group induced the need of a reliable animal model. The aim of the project is to identify a porcine cardiac cell population having similar characteristics as human CAP cells. Here a protocol for isolating cells from porcine heart biopsies have been established, as well as the in vitro expansion method. Additionally, the characterization of those expanded porcine cardiac cells was accomplished by growth kinetics study, FACS analysis, multilineage potential and myogenic induction assays. In this study we identified a not yet described porcine cardiac adherent cell population directly isolated from biopsies within the inner right ventricle. With almost $5\times {10}^{7}$ expanded cells after 30 days in culture, porcine cardiac cells present a high rate of proliferation in vitro. Porcine cardiac cells maintain a swirl-like pattern until 5-6 days in culture, and become fibroblast-like with a parallel organization when confluent. FACS analysis indicated that porcine cells are mainly CD90+ in opposite to human CAP cells. Both species show a similar CD45- and CD44+ profile. The multilineage assay showed no histological differentiation into fat, bone and cartilage on porcine cardiac cells like on human CAP cells. However gene expression analysis on porcine cardiac cells indicated that aP2 was upregulated in population undergoing an adipogenic induction, as well as osteopontine expression in osteogenic induction population. Porcine cardiac cell myogenic induction potential was tested on C2C12 myogenic mouse cells. The mouse cells differentiated in muscle in vitro thanks to the influence of those cardiac cells. Both cell populations have similar characteristics and the main distinction is the different CD90 expression profile. Those results encourage to realise additional studies, to confirm that those isolated cells could be a reliable model of human CAP cells.

In vitro modeling of complex microenvironmental regulation of stem cells

Yoji Tabata

Although stem cells hold tremendous potential for clinical applications, their in vitro manipulation remains very challenging. In vivo, stem cells reside in intricate 3D microenvironments, termed niche, in which many local and systemic extrinsic factors are integrated by the cells to induce controlled fate choices. Niche factors must not only be presented to stem cells in the correct composition, but also in a dynamic and spatially heterogeneous manner to evoke the appropriate response. However, state-of-the-art in vitro culture platforms fail to capture such microenvironmental complexity. In this thesis, a novel hydrogel-based microchip technology was invented to recapitulate in vitro the complexity of native stem cell niches. In this system, stem cells can be exposed to gradients of soluble or extracellular matrix (ECM)-tethered biomolecules. As a first proof of principle, this platform was used to locally influence the behavior of mouse embryonic stem cells (mESC) by exposing them to soluble, matrix-tethered or cell-secreted leukemia inhibitory factor (LIF). mESCs grown in synthetic 3D gels were found to respond to LIF gradients in a spatially regulated manner. In two subsequent studies, the microchip technology was utilized to recapitulate embryonic processes that are characterized by highly dynamic and spatially controlled morphogenetic processes that can currently not be modelled in a 3D context in vitro. First, an in vitro model of the developing central nervous system was conceived. In the developing vertebrate neural tube, dorsoventral identity of neural progenitors is defined by counter gradients of sonic hedgehog (Shh) and bone morphogenetic protein 7 (BMP7). Using the developing neural plate of chick embryos as a model system, a dedicated microchip was developed for exposing the neural progenitors in these tissues to gradients of Shh and BMP7. This approach allowed achieving, for the first time, ex vivo dorsoventral patterning. Secondly, a similar approach was employed towards modeling early human development in vitro. To this end, colonies of human embryonic stem cells (hESC) were exposed to opposing gradients of BMP4 and Noggin which induced mesendoderm differentiation exclusively in specific regions of the multicellular constructs. This study revealed the power of the developed platform to spatially control hESCs self-patterning. In a last set of experiments, the microchip technology was applied to mimic the complex molecular and cellular make-up of the bone marrow niche that regulates the activity of hematopoietic stem cells (HSC). A microchip system was successfully designed to capture the main anatomic features of native bone marrow niches with spatially separated compartments that supported either HSC dormancy or activation. Taken together, this thesis presents a set of innovative microengineering concepts for recapitulating the spatiotemporal complexity of native stem cell microenvironments. The platforms introduced here represent important additions to the currently rather limited set of in vitro tools for the study of complex multicellular phenomena in developmental biology, tissue engineering and regenerative medicine.

EPFL2017

Stem cell-based cardiovascular tissue engineering

Thomas Krähenbühl

Cardiovascular diseases, including myocardial infarction, are the leading cause of death worldwide for both men and women. Current therapies are limited by the restricted intrinsic regeneration capacity of the heart and by the lack of organs for transplantation. Human embryonic stem (hES) cells have the capacity to differentiate into various cell types, including vascular cells for forming de novo blood vessels to potentially repair the infarcted heart after myocardial infarction. Our goal was to improve cardiac function after myocardial infarction through hES-based cardiovascular tissue engineering. Our findings summarized: Improved cardiac performance, attenuated left ventricular dilation and decreased infarct size in infarcted rats can be attained after co-injection of hES-derived vascular cells and Thymosin β4 (Tβ4) in matrix metalloproteinase (MMP)-responsive hydrogels. Enhanced vascular cell adhesion, survival and organization can be achieved when coencapsulating bioactive peptide Tβ4 in MMP-responsive matrix in vitro. Directed cardiac differentiation of pluripotent cells can be influenced by matrix elasticity, adhesion ligand concentration and MMP-sensitivity in vitro. We first systematically modulated a three-dimensional (3D) MMP-responsive hydrogel, mimicking key biochemical characteristics of natural collagenous matrices, the major constituent of cardiac extracellular matrices. We found that these synthetic poly(ethylene glycol) (PEG)-based hydrogels can direct differentiation of pluripotent cardioprogenitors, using P19 embryonal carcinoma (EC) cells as a model, along a cardiac lineage in vitro. In order to systematically probe 3D matrix effects on P19 EC differentiation, matrix elasticity, MMP-sensitivity and the concentration of a matrix-bound RGDSP peptide, which is relevant in early cardiac developments were modulated. Soft matrices (E = 320 ± 64 Pa), mimicking the elasticity of embryonic cardiac tissue, increased the fraction of cells expressing the early cardiac transcription factor Nkx2.5 around 2-fold compared to embryoid bodies (EB) in suspension. In contrast, stiffer matrices (E = 4040 ± 420 Pa) decreased the number of Nkx2.5-positive cells significantly. Further indicators of cardiac maturation were promoted by ligation of integrins relevant in early cardiac development (α5β1, αvβ3) by the RGDSP ligand in combination with the MMP-sensitivity of the matrix, with a 6-fold increased amount of myosin heavy chain (MHC)-positive cells as compared to EB in suspension. This precisely controlled 3D culture system thus may serve as a potential alternative to natural matrices for engineering cardiac tissue structures for cell culture and potentially therapeutic applications. Neovascularization of infarcted tissue is a promising alternative strategy to potentially restoring lost contractile performance by cardiac cells. Vascular cells and cytokines are known to contribute to the protection of cardiomyocytes from death after myocardial infarction, but also in endogenous recruitment of vascular cells and even cardiomyocytes. The in vitro potential of the synthetic MMP-responsive PEG-hydrogel as a bioactive co-encapsulation system for vascular cells and a small bioactive peptide, Tβ4, was then examined. Tβ4 was previously shown to enhance survival of vascular cells and cardiomyocytes in ischemic environments, stimulate neovascularization after cardiac injury by inducing endogenous endothelial cell migration to the ischemic site, as well as play a key role in down-regulating expression of inflammatory molecules. We show that the physical incorporation of Tβ4 in this bioactive matrix creates a 3D environment conducive for human umbilical vein endothelial cell (HUVEC) adhesion, survival, migration and organization. Gels with entrapped Tβ4 increased the survival of HUVEC compared to gels without Tβ4 (p < 0.05), and significantly up-regulated the endothelial genes vascular endothelial-cadherin and angiopoietin-2, whereas the von Willebrand factor was significantly down-regulated (all p < 0.05). Incorporation of Tβ4 significantly increased MMP-2 and MMP-9 secretion of encapsulated HUVEC (p < 0.05). The gel acts as a controlled Tβ4-release system, as MMP-2 and MMP-9 enzymes trigger the release. In addition, Tβ4 facilitated HUVEC attachment and induced vascular-like network formation upon the PEG-hydrogels. These MMP-responsive PEG-hydrogels may thus serve as controlled co-encapsulation system of vascular cells and bioactive factors for in situ regeneration of ischemic tissues. Finally, we employed these injectable MMP-responsive bioactive hydrogels with vasculo-typic adhesion molecule as a provisional in situ reservoir of vascular cells derived from hES cells and Tβ4 peptide, in infarcted hearts. Typically, after myocardial infarction, the performance-generating contractile cardiac tissue is partially replaced by non-contractile fibrotic tissue, driven by upregulated enzymes such as MMP. Here, magnetic resonance imaging (MRI) revealed enhanced contractile performance 3 days and 6 weeks after myocardial infarction (both p < 0.001), attenuated left ventricular dilation (p < 0.05) and decreased infarct size at 6 weeks (p < 0.05) as compared to infarcted rats with PBS injection. . We found the gel substituting the native degrading extracellular matrix in the infarcted myocardium and promoting structural organization of endothelial cells and cardiomyocytes, while the delivered hES-derived vascular cells formed de novo capillaries in the infarct zone.

EPFL2009

Early Fate Choice Determines in vivo Engraftment and in vitro Behavior of Epidermal Stem Cells

François Gorostidi

Regenerative medicine aims at using stem cells to restore or establish lost, damaged, diseased, or aging tissues and organs function. The transplantation of cultured epidermal autograft (CEA) has saved the lives of many burned patients over the last 30 years, but the resulting epidermis is far from perfect, and clinical outcomes remain unpredictable. The engraftment of cultured epidermal stem cells is poorly understood, and, as a result, suboptimal. Using a large animal model, the pig, we recapitulated the clinical settings and outcomes of human CEA transplantation. With this model, we study the engraftment process, and we demonstrate that cultured epidermal stem cells often, but not always, favor the choice of differentiation over self-renewal when transplanted on full thickness wounds. Differences in this early fate choice are likely to explain the variability of clinical outcomes. We developed a system of in vitro live cell imaging (LCI) to study the fate choices of multiple individual cells in a microenvironment that is much more controlled than the grafting bed of a burned patient. We demonstrate that keratinocytes have a very heterogeneous behavior in vitro. With a fluorescent reporter of cell cycle progression (FUCCI), we show the influence of the cell cycle on early fate choices of cultured keratinocytes. By introducing a ROCK inhibitor in the culture medium, we show that keratinocyte’s early fate choices can be impacted so that they show greater clonogenicity and growth potential. The unique combination of large animal CEA transplantation with individual cell LCI demon- strate the critical role of early fate choice, both for epidermal stem cell engraftment and in vitro behavior. The possibility to influence the early fate choices of stem cells by modifying the microenvironment has a great potential to improve the engraftment and the ex vivo ampli- fication of stem cells, the two limiting steps of cell replacement therapy.

EPFL2012