Innovative sources of neural crest - derived ectomesenchymal stem cells for hepatic bioengineering

Abstract

Liver disorders affect more than 100 million people worldwide and the therapeutic approaches for severe forms such as fulminant toxic hepatitis, cirrhosis, cancer and autoimmune liver disease remain palliative, awaiting for an organ transplant from a donor. An innovative approach involves intrahepatic transplantation of adult, multipotent stem cells of extrahepatic origin, whose up to now the mostly widely used have been mesenchymal stromal cells (MSC) from the bone marrow (bm-MSC). Specifically, MSC have been preliminarily in vitro or post-transplant in vivo induced to hepatocyte differentiation, and then associated with a biocompatible heterologous three-dimensional (3D) support like a decellularized or biostamped collagen matrix. Indeed, the 3D geometry of the matrix is believed to regulate the 3D self-assembly and differentiation of hepatic progenitors, as predicted by Gerald Edelman's Topobiological Theory. Since MSC from bone marrow and other sources exhibit a number of limitations in hepatic differentiation, the aim of my study has been the identify and evaluate innovative sources of adult stem cells suitable for in vitro and ex situ (i.e. once used for a 3D matrix recellularization) hepatic differentiation. My interest has been focussed on the still poorly investigated differentiation potential of adult ectomesenchymal cells of neural crest origin (i.e. adult neuroectodermal stem cells), like those expected to be found in the adult thyroid gland and the cartilage of the nasal septum. In particular, recent evidence collected by the group of research leaded by Prof. Roberto Toni at the University of Parma, in Italy has shown that adult stem cells can in vitro be isolated and expanded using long-term (2-4 months) primary cultures from the male rat thyroid. Preliminary data obtained in collaboration with Prof. Stephen Pennington of the UCD – Ireland on their proteomic signature have shown a very high expression of mesenchymal and, to a lesser extent endodermal protein markers. However, a minor fraction of these markers also includes proteins from a neuroectodermal lineage, in agreement with very recent evidence that the neural crest contribute to the development of the thyroid gland giving rise to its stromal matrix and, possibly to other still unknown progenitor cells. Since these cultures of adult rat stem cells can be in vitro differentiated to functional thyrocytes (that are classical endodermal cells like the hepatocytes), I reasoned that they could represent an innovative model source for hepatic differentiation. To reach this goal, in the first year of my PhD program I preliminary widened the previously acquired data on the proteomic signature of our rat thyroid stem cells / progenitors (TSC/P) using an LC-MS/MS SRM technique based on in silico sequences of not previously investigated proteins of endodermal, mesodermal, and neuroectodermal lineage as well as markers of epithelial-mesenchymal transition. A semiquantitative evaluation based on score values depicted an increase in the number of markers of neuroectodermal lineage, and an evidence of some proteins of epithelial-mesenchymal transition. These results support the original assumption of our group that a large fraction of our rat TSC/P are ectomesenchymal cells of neural crest origin, although a minor effect on the induction of a mesenchymal phenotype was triggered by the monolayer culture per se, as suggested by the presence of few markers of epithelial-mesenchymal transition. In addition, using light microscopic histochemistry, immunocytochemistry, and scanning electron microscopy I evaluated the hepatic differentiation of our rat TSC/P tested with two protocols of in vitro differentiation, both originally developed in our lab. at UNIPR. Of these, only one (n.1) was able to induce a satisfactory hepatocyte differentiation. A key role in this result was likely played by the use of Hepatocyte Growth Factor (HGF), whose Met receptor is expressed in neural crest and neural crest- derived cells like the ectomesenchyme, and also in differentiated hepatocytes and thyrocytes suggesting a great affinity of our TSC/P for an hepatic lineage. As a comparison, in the second year of my PhD program I studied a similar differentiation procedure using the currently believed, cellular gold standard for hepatic differentiation i.e. adult male rat bm-MSCs. Based on the same technologies as during the first year of work but in contrast to TSC/P, none of the two differentiation protocols used provided in vitro satisfactory development of bm-MSCs to hepatocytes. Since HGF may result in downregulation of MSC differentiation to hepatocytes when administered for long periods of times (as in our models), and use of Epidermal Growth Factor should be delivered at periods earlier than those chosen in our protocols, it is concluded that the weak differentiation results obtained depend on differentiation protocols for MSC not efficient enough when their multipotentiality is lowered by repeated culture passages from P3 ahead. In the third year of my PhD program, I substantiated the results on the proteomic signature of TSC/P and bm-MSC following their hepatic differentiation using qualitative LC-LIT-Orbitrap XL. Remarkably, hepatic differentiation of TSC/P with the protocol that had shown the best immunocytochemical performances during the first year of work (n.1) confirmed upregulation of the hepatic marker albumin, and revealed presence of S100-A6, a protein associated with activated hepatic stellate cells. Since the latter are a normal component of the liver in vivo, and are of mesodermal origin, the present in vitro evidence supports the original assumption that the large majority of our TSC/P are cell from an ectomesenchymal, neural-crest derived lineage easily driven to hepatocytes once triggered with adequate differentiation factors. In contrast, MSC differentiated with both previous protocols exhibited a proteomic profile with numerous common markers, confirming their immaturity and resistance to hepatic differentiation in the experimental setting applied. Finally, in search for a new source of neural crest-derived, ectomesenchymal cells potentially suitable for hepatic differentiation in humans (as those found in the rat and previously described), I engaged myself in the detailed lineage and molecular characterization of a very peculiar type of adult stem cells, obtained from the cartilage of the human nasal septum. This work has been developed between 2017 and 2018 under the tenure of my PhD Mobility Program, and in collaboration with the group of Prof. Ivan Martin at the Laboratory of Tissue Engineering / Department of Surgery and Biomedicine of the University Hospital in Basel, Switzerland. Prof. Martin and his group originally studied the gene expression of these cells, and used them as a new mean for repair of articular cartilage defects. They are adult nasal chondrocytes, taken following in vivo surgical sampling, and then in vitro de-differentiated. Recent collaborative studies between the groups of Prof. Toni and Prof. Martin have shown that a number of neuroectodermal markers of these cells can be found in the nasal septum of the human embryo, and can be recognized in the adult nasal chondrocytes by flow cytometry. During this last year of my PhD and using immunocytochemistry, I have evaluated the distribution of the majority of those embryonic markers also in primary cultures of adult nasal chondrocytes. In addition, using statistical analysis I have re-analyzed and re-calculated the quantitative results previously obtained by flow cytometry in nasal chondrocytes on the distribution of markers of mesodermal and neuroectodermal origin. Finally, using qualitative LC-LIT-Orbitrap XL I have obtained a preliminary profile of these adult stem cells showing a molecular setting based on downregulation of glycolytic enzymes and some DNA-associated histones as opposed to upregulation of a number of proteins involved in morphogenetic and developmental processes. Collectively, these data suggest that nasal chondrocytes are prone to a high plasticity of their phenotype, as expected for multipotent stem cells. Since flow cytometry showed that more than 80% of them express cytoplasmic and membrane markers consistent with ectomesenchymal neural crest-derived cells, the current proteomic profile (although still incomplete) supports the original hypothesis that they might successfully be induced to differentiate to cell lineages similar to those observed with ectomesenchymal, neural crest-derived rat thyroid stem cells. As such, I expect that these adult human stem cells may represent a new source for differentiation to human hepatocytes, and become a very innovative tool for regenerative medicine and tissue engineering in disorders of the human liver.