Abstract:
A fundamental question in developmental and stem cell biology is how asymmetric divisions of
stem/progenitor cells are coordinated to give rise to two distinct daughter cells. One strategy that has been
employed for model development exploits the fact that a local and immobilized Wnt3a signal can
coordinate asymmetric divisions of mouse pluripotent stem cells in vitro. Unfortunately, this method is
currently limited due to the random nature of the culture techniques it employs, making in-depth molecular
and cellular analysis impossible. Furthermore, the ability of cells other than mouse pluripotent cells to
undergo asymmetric divisions under these conditions remains unknown. In order to address these issues,
we have developed an accessible 3D bioprinting platform that allows for precise placement of cells and
growth factors. By bioprinting coordinated grids of growth-factor coated microbeads and stem cells into
thousands of precise locations, our system ensures single cell and microbead interactions. This results in
a 1000-fold increase in the efficiency of the process allowing for robust, high-throughput analysis. Here we
propose to adapt this technology to further explore the capacity of localized signals to drive asymmetric
divisions, and to probe the role of specific epigenetic mediators in the process. Therefore, Aim 1 will
determine if human pluripotent and mouse/human multipotent stem cells undergo asymmetric divisions in
the presence of a localized self-renewal signal. In Aim 2, we will explore differences in active
demethylation regulators between daughter cells of mouse pluripotent cell asymmetric divisions. Active
demethylation is a critical point of focus because 5-hydroxymethylcytosine (5hmC) has been reported to
segregate unevenly during asymmetric divisions. Additionally, we will examine the active demethylation
agent ten-eleven translocation methylcytosine dioxygenase 1 (TET1), and TET1 regulatory micro-RNA
family 29 (miR-29) due the their involvement in pluripotency, self-renewal, and early differentiation.
Together, these aims will test the hypothesis that localized signaling molecules direct asymmetric divisions
that result in daughter cells with different levels of the active demethylation agents 5hmC, TET1, and miR-
29. Upon completion of this study, we will have characterized various cellular responses to localized
signaling, and evaluated a potential molecular mediator of the process. Furthermore, due to the inherent
nature of our computer numerical controlled system, we are able to faithfully disseminate the protocols
digitally for repeating our experiments to other laboratories. Thus, our proposal will also establish an
experimental foundation that will support future complex molecular analysis of asymmetric divisions in the
scientific community.
