Karolien Buyl

Mesoderm-Derived Stem Cells: The Link Between the Transcriptome and Their Differentiation Potential

Human adult stem cells (hASCs) have become an attractive source for autologous cell transplantation, tissue engineering, developmental biology, and the generation of human-based alternative in vitro models. Among the 3 germ cell layers, the mesoderm is the origin of todays most widely used and characterized hASC populations. A variety of isolated nonhematopoietic mesoderm-derived stem cell populations exist, and all of them show important differences in terms of function, efficacy, and differentiation potential both in vivo and in vitro. To better understand whether the intrinsic properties of these cells contribute to the overall differentiation potential of hASCs, we compared the global gene expression profiles of 4 mesoderm-derived stem cell populations: human adipose tissue-derived stromal cells, human bone marrow-derived stromal cells (hBMSCs), human (fore)skin-derived precursor cells (hSKPs), and human Whartons jelly-derived mesenchymal stem cells (hWJs). Significant differences in gene expression profiles were detected between distinct stem cell types. hSKPs predominantly expressed genes involved in neurogenesis, skin, and bone development, whereas hWJs and, to some extent, hBMSCs showed an increased expression of genes involved in cardiovascular and liver development. Interestingly, the observed differential gene expression of distinct hASCs could be linked to existing differentiation data in which hASCs were differentiated toward specific cell types. As such, our data suggest that the intrinsic gene expression of the undifferentiated stem cells has an important impact on their overall differentiation potential as well as their application in stem cell-based research. Yet, the factors that define these intrinsic properties remain to be determined.

Characterization of hepatic markers in human Wharton’s Jelly-derived mesenchymal stem cells

Stem cell technology could offer a unique tool to develop human-based in vitro liver models that are applicable for testing of potential liver toxicity early during drug development. In this context, recent research has indicated that human Whartons Jelly-derived mesenchymal stem cells (hWJs) represent an interesting stem cell population to develop human hepatocyte-like cells. Here, an in-depth analysis of the expression of liver-specific transcription factors and other key hepatic markers in hWJs is evaluated at both the mRNA and protein level. Our results reveal that transcription factors that are mandatory to acquire and maintain an adult hepatic phenotype (HNF4A and HNF1A), as well as adult hepatic markers (ALB, CX32, CYP1A1, CYP1A2, CYP2B6 and CYP3A4) are not expressed in hWJs with the exception of K18. On the contrary, transcription factors involved in liver development (GATA4, GATA6, SOX9 and SOX17) and liver progenitor markers (DKK1, DPP4, DSG2, CX43 and K19) were found to be highly expressed in hWJs. These findings provide additional indication that hWJs could be a promising stem cell source to generate hepatocyte-like cells necessary for the development of a functional human-based in vitro liver model.

Evaluation of a new standardized enzymatic isolation protocol for human umbilical cord-derived stem cells.

The umbilical cord (UC) represents an important source of mesenchymal stem cells (MSC). These human UC-derived MSC (UC-MSC) have already been isolated using a protocol based on the migratory and plastic adhesive properties of MSC (UC-MSC-Mig). The UC-MSC-Mig isolation method, however, is difficult to standardize. Therefore, we developed an enzymatic isolation protocol (UC-MSC-Enz) to overcome the above mentioned disadvantages. First, we investigated the UC-MSC-Enz for their MSC properties. We found that UC-MSC-Enz express the MSC markers CD73, CD90 and CD105 and are able to differentiate into osteoblasts, adipocytes and chondroblasts fulfilling the MSC criteria of the International Society for Cellular Therapy. Previously we found that UC-MSC-Mig are unique among MSCs due to their significant expression of several hepatic (progenitor) markers. Therefore, we also investigated the expression of hepatic transcription factors and other hepatic markers in UC-MSC-Enz at both the mRNA and protein level. We found that the expression of hepatic transcription factors (GATA4, GATA6, SOX9 and SOX17) and hepatic markers (AFP, DPP4, CX43, DKK1, DSG2, KRT18 and KRT19) in UC-MSC-Enz was not significantly different from those of UC-MSC-Mig. Consequently, this optimized enzyme-based method represents a fast, robust and standardized way to isolate UC-MSC for a broad range of applications.

Measurement of Albumin Secretion as Functionality Test in Primary Hepatocyte Cultures.

One of the most important functions of hepatocytes is the synthesis of serum proteins, more specifically of serum albumin. Albumin secretion in serum is essential, since it maintains the oncotic pressure in the body. Measurement of albumin secretion is used as a liver function test to indicate potential liver injury and liver pathology. In this chapter, a protocol for the measurement of albumin secretion in the supernatant of cultured rat hepatocytes is described. The procedure relies on an enzyme-linked immunosorbent assay allowing rat albumin to be quantitatively measured.

Human-based systems: Mechanistic NASH modelling just around the corner?

Graphical abstract Figure. No caption available. &NA; Non‐alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by excessive triglyceride accumulation in the liver accompanied by inflammation, cell stress and apoptosis. It is the tipping point to the life‐threatening stages of non‐alcoholic fatty liver disease (NAFLD). Despite the high prevalence of NASH, up to five percent of the global population, there are currently no approved drugs to treat this disease. Animal models, mostly based on specific diets and genetic modifications, are often employed in anti‐NASH drug development. However, due to interspecies differences and artificial pathogenic conditions, they do not represent the human situation accurately and are inadequate for testing the efficacy and safety of potential new drugs. Human‐based in vitro models provide a more legitimate representation of the human NASH pathophysiology and can be used to investigate the dysregulation of cellular functions associated with the disease. Also in silico methodologies and pathway‐based approaches using human datasets, may contribute to a more accurate representation of NASH, thereby facilitating the quest for new anti‐NASH drugs. In this review, we describe the molecular components of NASH and how human‐based tools can contribute to unraveling the pathogenesis of this disease and be used in anti‐NASH drug development. We also propose a roadmap for the development and application of human‐based approaches for future investigation of NASH.

Human Skin-Derived Precursor Cells: Isolation, Expansion, and Hepatic Differentiation.

Human skin-derived precursor cells are a multipotent stem cell population that resides within the dermis throughout adulthood. Human skin-derived precursor cells can be isolated, purified, and expanded in large quantities from any patient, in health and disease, and differentiated to mesodermal and ectodermal cell types. Recently, it was also found that they can be directed towards hepatic cells with acquired properties of toxicological relevance. As such, they represent a valuable cell source for the further development of human-relevant in vitro models for the identification and quantification of hepatotoxic compounds. In this chapter, a robust basic methodology to isolate, expand, and differentiate human skin-derived precursor cells into hepatic cells in a sequential and time-dependent way is provided.