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.
Understanding the basic mechanisms behind how stem cells develop into different cell types is critical for the generation of new therapies for cancer, tissue repair, and developmental disorders. This proposal will use newly developed three-dimensional bioprinting technology to advance our understanding of this issue by carefully examining how stem cells confer different information to daughter cells during divisions.
