Genetic modification of human embryonic stem cells for lineage selection, derivation and analyses of human 3rd pharyngeal pouch epithelium like cells and its derivatives
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Date
07/07/2017Item status
Restricted AccessEmbargo end date
31/12/2100Author
Kaushik, Suresh Kumar
Metadata
Abstract
Human pluripotent stem cells (hPSCs) such as, human embryonic stem cells
(hES) and human induced pluripotent stem cells (hiPS) are a valuable
resource to generate bespoke cell types for a number of therapeutic
applications involving cell therapy, drug screening and disease modelling.
The overarching goal of this project was to generate a set of transgenic tools
by gene targeting and genetic modification of hESCs for applications in stem
cell biology such as the in vitro isolation, analyses and derivation of lineage
specific cell types. The transgenic tools generated in this study were
designed and tested in particular for the human 3rd pharyngeal pouch
epithelium (3PPE) like cells and its derivatives, namely the thymus and
parathyroid, which are key organs involved in T-cell development and
calcium homeostasis respectively.
The forkhead transcription factor FOXN1 is considered a master regulator of
the development of the thymic epithelium (TEC), the major functional
component of the thymic stroma, which is intimately involved in T-cell
differentiation. So, to facilitate the prospective isolation of FOXN1 expressing
TECs, gene targeting was employed to place a fluorescent reporter and a
lineage selection antibiotic resistance gene under the direct control of the
endogenous FOXN1 promoter. To date, I have not been able to detect either
the fluorescent reporter, or FOXN1 expression using published directed
differentiation protocols, but only what can be deemed as precursors
expressing the cytokeratin K5 and other markers associated with the
development of the thymus and parthyroid from 3PPE. The lack of
endogenous FOXN1 activation was observed in both the unmodified parent
and the targeted FOXN1 knock-in human ES lines. Further, over-expression
of FOXN1 cDNA during the differentiation protocol did not result in the
activation of endogenous FOXN1. So, the results evinced in this study could
be due to a number of reasons such as, technical issues associated with
transference of the published protocols to the cell lines used in this study,
differences in hESC lines, and effects of different hESC culture methods and
practices.
The homeobox gene HOXA3 is expressed in the 3PPE during development.
So, a HOXA3 transgenic reporter hESC line could be an invaluable tool for
prospective isolation of in vitro derived 3PPE like cells. The reporter was
generated by Piggy Bac transposase mediated transposition of a HOXA3
containing Bacterial Artificial Chromsome (BAC) in the FOXN1 knock-in
human ES line. To date, this is biggest reported cargo that has been
successfully transposed in human ESCs. Moreover, this is the first lineage
specific double reporter transgenic hESC line that has been reported for this
lineage. This HOXA3 reporter line was then used to isolate and enrich for
HOXA3 expressing 3PPE like cells with very high efficiencies during the
directed differentiation of hESCs, thus demonstrating the key objective of
this transgenic hESC line for this study.
In a novel parallel approach, I have conceived, designed and generated
transgenic hESCs lines capable of inducible and constitutive over-expression
of key transcription factors involved in the development of 3PPE and its
derivatives, the thymus and parathyroid. The objective of the said over-expression
hESC lines was to interrogate if such a system could elicit
morphological and gene expression changes in hESCs following over-expression.
By testing the chosen panel of transcription factors in hESCs, I
was able to detect cells expressing FOXN1 and GCMB, which are key
markers of TECs and PTECs. Further, I have isolated an expandable
population of cells expressing markers analogous to their in vivo counterpart
found in the 3PPE of a developing mouse embryo around E9.0. The in vivo
potency of these in vitro derived 3PPE like cells is yet to be ascertained.
Nevertheless, transgenic constructs generated in this experiment could also
be tested during future attempts at the differentiation of hESCs to TECs and
PTECs, and also used as a basis for future studies involving the direct
conversion of patient specific fibroblasts to 3PPE like cells and its derivatives.
In summary, several transgenic tools developed in this project, namely the
FOXN1 knock-in transgenic hESC line, FOXN1-HOXA3 double transgenic
hESC line, over-expression 3PPE transgenes and hESC transgenic lines, and
results from the deployment of these tools provide a foundation, from which
protocols to generate functional TECs and PTECs can be refined and
optimised. These transgenic hESC lines also provide a tractable model, which
could be used to interrogate the development of human TECs and PTECs
from human 3PPE, and identify hitherto unknown early events in their
development in an in vitro reductionist setting.