4711795_monash_158654.pdf (75.58 MB)
Download file

Directed derivation and FACS-mediated purification of PAX3+/PAX7+ skeletal muscle precursors from human pluripotent stem cells

Download (75.58 MB)
thesis
posted on 02.03.2017, 00:46 authored by Borchin, Bianca
Human pluripotent stem cells (hPSCs) constitute a promising resource for use in cell-based therapies and a valuable in vitro model for studying early human development and disease. Despite significant advancements in the derivation of specific fates from hPSCs, the generation of skeletal muscle remains challenging and is mostly dependent on transgene expression. Here, we describe a method based on the use of a small-molecule GSK3β inhibitor to derive skeletal muscle from several hPSC lines. We show that early GSK3β inhibition is sufficient to create the conditions necessary for highly effective derivation of muscle cells. Moreover, we developed a strategy for stringent fluorescence-activated cell sorting-based purification of emerging PAX3+/PAX7+ muscle precursors that are able to differentiate in postsort cultures into mature myocytes. This transgene-free, efficient protocol provides an essential tool for producing myogenic cells for in vivo preclinical studies, in vitro screenings, and disease modeling.

History

Principal supervisor

Jose M. Polo

Year of Award

2015

Department, School or Centre

Australian Regenerative Medicine Institute (ARMI)

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences