Directed differentiation

Directed differentiation is a bioengineering methodology at the interface of stem cell biology, developmental biology and tissue engineering. It is essentially harnessing the potential of stem cells by constraining their differentiation in vitro toward a specific cell type or tissue of interest. Stem cells are by definition pluripotent, able to differentiate into several cell types such as neurons, cardiomyocytes, hepatocytes, etc. Efficient directed differentiation requires a detailed understanding of the lineage and cell fate decision, often provided by developmental biology. Directed differentiation is a bioengineering methodology at the interface of stem cell biology, developmental biology and tissue engineering. It is essentially harnessing the potential of stem cells by constraining their differentiation in vitro toward a specific cell type or tissue of interest. Stem cells are by definition pluripotent, able to differentiate into several cell types such as neurons, cardiomyocytes, hepatocytes, etc. Efficient directed differentiation requires a detailed understanding of the lineage and cell fate decision, often provided by developmental biology. During differentiation, pluripotent cells make a number of developmental decisions to generate first the three germ layers (ectoderm, mesoderm and endoderm) of the embryo and intermediate progenitors, followed by subsequent decisions or check points, giving rise to all the body's mature tissues. The differentiation process can be modeled as sequence of binary decisions based on probabilistic or stochastic models. Developmental biology and embryology provides the basic knowledge of the cell types' differentiation through mutation analysis, lineage tracing, embryo micro-manipulation and gene expression studies. Cell differentiation and tissue organogenesis involve a limited set of developmental signaling pathways. It is thus possible to direct cell fate by controlling cell decisions through extracellular signaling, mimicking developmental signals. Directed differentiation is primarily applied to pluripotent stem cells (PSCs) of mammalian origin, in particular mouse and human cells for biomedical research applications. Since the discovery of embryonic stem (ES) cells (1981) and induced pluripotent stem (iPS) cells (2006), source material is potentially unlimited.Historically, embryonic carcinoma (EC) cells have also been used. Fibroblasts or other differentiated cell types have been used for direct reprogramming strategies. Cell differentiation involves a transition from a proliferative mode toward differentiation mode. Directed differentiation consists in mimicking developmental (embryo's development) decisions in vitro using the stem cells as source material. For this purpose, pluripotent stem cells (PSCs) are cultured in controlled conditions involving specific substrate or extracellular matrices promoting cell adhesion and differentiation, and define culture media compositions. A limited number of signaling factors such as growth factors or small molecules, controlling cell differentiation, is applied sequentially or in a combinatorial manner, at varying dosage and exposure time. Proper differentiation of the cell type of interest is verified by analyzing cell type specific markers, gene expression profile, and functional assays. support cells and matrices provide developmental-like environmental signals. This method consists in exposing the cells to specific signaling pathways modulators and manipulating cell culture conditions (environmental or exogenous) to mimick the natural sequence of developmental decisions to produce a given cell type/tissue. A drawback of this approach is the necessity to have a good understanding of how thecell type of interest is formed. This method, also known as transdifferentiation or direct conversion, consists in overexpressing one or several factors, usually transcription factors, introduced in the cells. The starting material can be either pluripotent stem cells (PSCs), or either differentiated cell type such as fibroblasts. The principle was first demonstrated in 1987 with the myogenic factors MyoD.A drawback of this approach is the introduction of foreign nucleic acid in the cells and the forced expression of transcription factors which effects are not fully understood. This methods consists in selecting the cell type of interest, usually with antibiotic resistance. For this purpose, the source material cells are modified to contain antibiotic resistance cassette under a target cell type specific promoter. Only cells committed to the lineage of interest is surviving the selection. Directed differentiation provides a potentially unlimited and manipulable source of cell and tissues.Some applications are impaired by the immature phenotype of the pluripotent stem cells (PSCs)-derived cell type, which limits the physiological and functional studies possible.Several application domains emerged: