The directed differentiation of human pluripotent cells to specified lineages in vitro follows many of the tenets established in developmental biology. The mechanisms and basic principles established in embryology have been carried over to in vitro systems and adapted for designing protocols to differentiate mature cell types from pluripotent cells. Conditions for growing ESCs have been established to mimic the developmental conditions that either maintain pluripotency or promote differentiation to specific lineages. However, cell culture is inherently unlike development; cells are exposed to vastly different selective conditions, and techniques for differentiation are designed to purify or enrich for specific cell types rather than reproducing all the interacting cell types within an embryo or tissue. Therefore, the criteria used to assess differentiation are distinct from those used in development, and these systems should not be treated analogously. In addition, the signaling that occurs to specify lineage fate during development is extremely complex and not fully understood, and therefore cannot be reproduced using in vitro systems.
Cell types differentiated from pluripotent cells are characterized based on marker ex
Following the guidelines established in embryology, it has been possible to induce the formation of ectoderm, mesoderm, and endoderm lineages, as well as many of their downstream derivatives. The temporal and spatial segregation of cells observed during gastrulation and subsequent developmental stages implies that divergences in cell fate are based in part on a range of diverse signaling gradients to induce specific lineages. Using ESC-based techniques, it is possible to determine the effect of specific growth factors on development of lineages in a highly-controlled environment, and observe whether the efficiency of differentiation to specified lineages can be linked to concentration-dependent or temporal-dependent effects.
There are two main methods that have been developed to differentiate ESC and iPSC, and each has benefits and limitations. In the first method, differentiation can be accomplished by formation of embryoid bodies (EBs) in which cells are aggregated in suspension, whereupon they differentiate towards all 3 germ layers in a way that is reminiscent of embryogenesis. Growth factors and media conditions can be altered within this environment to enhance differentiation of specific cell types. Following a specified period of time, EBs are allowed to attach in culture and cells of interest can be selected based on growth conditions, morphological criteria, or FACS sorted from this population based on a set of surface markers.
Alternatively, pluripotent cells can be differentiated by plating onto a supportive substrate such as Matrigel, or onto a feeder layer substrate to allow some degree of cell-cell or cell-matrix interaction. These methods provide a more controlled environment to select specific cell types, and monitor cell differentiation towards a desired germ layer or cell type. However, this method also requires more precise control of cell culture conditions, including the substrate or feeder cell type, the addition of exogenous growth factors, and temporal control during the course of differentiation. Similar to EB methods, protocols can be fine-tuned based on morphology or protein marker ex
Many of the growth factor cues in stem cell differentiation are implemented by a small number of signals, including Wnt, Nodal and BMP, which are dynamically coordinated during development to define multiple cell types. Manipulation of these regulatory pathways in vitro using ESCs has revealed that they have dramatic effects on the regulation of germ layer development in human cells as well. For instance, activation of the Nodal pathway using Activin A induces formation of endoderm and mesoderm, in a concentration-dependent manner. Conversely, early ectodermal specification has been shown to be induced by BMP signaling. Later in differentiation, BMP-4 inhibits neuronal differentiation and consequently promotes selection of definitive ectoderm or epidermal lineages. However, often the same conditions can lead to different results depending on cellular target. Since cells at the early stages of differentiation are still uncommitted and therefore unstable, even an accurate reproduction of the microenvironmental conditions may result in a heterogeneous population of cells. BMP-4, for example, has been shown in different studies to induce endodermally-derived hepatocytes, mesoderm-derived hemangioblasts, and ectodermally-derived keratinocytes. To achieve effective and specific differentiation, the aggregate sum of environmental conditions needs to be considered, with concentration, spatial and temporal control. Thus, by altering the substrate conditions and growth-factor supplements, it is possible to select specific germ layers in early differentiation, and then further select for or against other lineages at later stages of differentiation. The result is the establishment of a series of defined protocols that have been established to differentiate specified cell types from pluripotent sources. Since this thesis is focused on the differentiation of cells towards skin-related cell types, only the methods relevant to those cells are described here.