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A platform for the differentiation of human pluripotent stem cells

thesis
posted on 16.02.2017, 03:06 by Ng, Elizabeth Siew-Sun
The successful isolation and in vitro culture of human embryonic stem (ES) cells in 1998 by James Thomson ushered in a new era in biology. It was immediately realised that human ES cells might provide a potentially unlimited source of normal human cells for therapeutic use, if they could be differentiated to yield products that were functional and safe. The aim of this project was to adapt methods used for differentiation of mouse ES cells to the new field of human ES cells. My initial studies explored the role of the homeobox gene, Mix/1, in the development of haematopoietic mesoderm in differentiating mouse ES cells. These experiments, using Mix/1 reporter ES cell lines, showed that Mix/1 was required for efficient differentiation of cells from the primitive streak stage to blood. Furthermore, the use of a serum-free medium enabled me to demonstrate that the growth factors bone morphogenetic protein (BMP)4 or Activin A were required for the induction of Mix/1 and subsequent haematopoietic patterning of mesoderm. Attempts to differentiate human ES cells as embryoid bodies (EBs) initiated from clumps of cells were hampered by variable differentiation outcomes, probably due to variation in the size of the ES cell clumps. In order to generate more uniform EBs, we developed a method in which enzymatically dispersed human ES cells were reaggregated to form spherical cellular masses, termed 'spin EBs'. In a serum-free differentiation medium supplemented with growth factors, the spin EBs differentiated efficiently and synchronously, as evidenced by the sequential expression of molecular markers representing stem cells, primitive streak, and mesoderm. The differentiated cells could be analyzed by flow cytometry, sorted for further culture or used for gene expression analyses. In subsequent studies, the formulation of the differentiation medium was modified to devise a base medium that included only three recombinant human proteins and was free of animal products, denoted APEL. The integration of APEL medium with the spin EB method resulted in a robust platform for growth factor-directed human ES cell differentiation that has been used by our laboratory and others to support differentiation to mesoderm, endoderm and ectoderm. The final chapters of this thesis report studies using the spin EB method with APEL medium to characterize early human ES cell differentiation. Analogous to our earlier mouse ES cell experiments, we generated and characterized human MIXL1 reporter ES cells. We demonstrated that human MIXL1 was also transiently expressed in response to BMP4 or ACTIVIN A stimulation. These experiments demonstrated the utility of the MIXL1GFPfw human ES cell reporter line for analyzing the previously inaccessible events surrounding the development of human primitive streak-like cells and their subsequent commitment to haematopoiesis. In the final series of experiments, we reported that vibrational spectroscopy, a modality that generates infrared spectra that reflect the macromolecular composition of cells, could distinguish undifferentiated human ES cells from early mesendoderm or ectoderm. These findings suggested that spectroscopic analysis might provide objective indicators of sternness and differentiation that could complement conventional morphological, flow cytometric and gene expression markers.

History

Principal supervisor

Edouard Stanley

Year of Award

2014

Department, School or Centre

Anatomy and Development Biology

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences